Diagnosis and treatment of autoimmune diseases

ABSTRACT

Methods, kits and compositions for diagnosing and treating autoimmue diseases such as rheumatiodi arthritis, Crohn&#39;s disease, and ulcerative colitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/030,790, filed on Apr. 20, 2016, now U.S. Pat. No. 10,101,344,which is a national stage filing under 35 U.S.C. § 371 of internationalPCT application PCT/US2014/061242, filed on Oct. 17, 2014, which claimspriority to U.S. Provisional Application Ser. No. 61/893,542, filed onOct. 21, 2013, the entire contents of each of which are incorporated byreference herein.

BACKGROUND

Plasma kallikrein (pKal) is the primary bradykinin-generating enzyme inthe circulation and a component of the plasma kallikrein-kinin system(KKS). Colman, R. W., and Schmaier, A. H. (1997) Blood 90, 3819-3843.The activation of pKal occurs via the contact system which has beendemonstrated to be causative in the disease pathology associated withhereditary angioedema (HAE). Zuraw, B. L., and Christiansen, S. C.(2008) Expert Opin Investig Drugs 17, 697-706. Bradykinin is a keymediator of pain, inflammation, edema and angiogenesis. Maurer, M., etal. (2011) Allergy 66, 1397-1406; Colman, R. W. (2006) Curr Pharm Des12, 2599-2607.

SUMMARY

The present disclosure is based on the observations that the levels ofcleaved high molecular weight kininogen (HMWK) are elevated in patientshaving an autoimmune disease, such as rheumatoid arthritis (RA), Crohn'sdisease (CD), and ulcerative colitis (UC). Accordingly, disclosed hereinare methods for diagnosing an autoimmune disease such as RA, CD or UC,monitoring progress of the autoimmune disease, or assessing efficacy ofa treatment for the autoimmune disease based on the level of cleavedHMWK.

In one aspect, the present disclosure provides a method of diagnosing anautoimmune disease (e.g., RA, CD, or UC) in a subject, the methodcomprising: (i) providing a biological sample (e.g., a serum sample or aplasma sample) of a subject suspected of having the autoimmune disease;(ii) measuring a level of cleaved high molecular weight kininogen (HMWK)in the biological sample; and (iii) identifying the subject as having orat risk for the autoimmune disease, if the level of cleaved HMWK in thebiosample is elevated as compared to a control sample.

In some embodiments, the level of cleaved HMWK is measured by an assaythat involves a binding agent (e.g., an antibody) specific to cleavedHMWK. The assay can be an enzyme-linked immunosorbent assay (ELISA) oran immunoblotting assay, e.g., a Westernblotting assay involving LiCordetection.

The method can further comprise subjecting the subject to a treatmentfor the autoimmune disease. In some embodiments, the subject isadministered with an effective amount of a plasma kallikrein (pKal)inhibitor, e.g., those described herein.

In another aspect, the present disclosure provides a method ofmonitoring development of an autoimmune disease (e.g., RA. CD, or UC) ina subject, the method comprising: (i) providing a first biologicalsample of a subject suspected of having the autoimmune disease at afirst time point; (ii) measuring a first level of high molecular weightkininogen (HMWK) in the first biological sample; (iii) providing asecond biological sample of the subject at a second time pointsubsequent to the first time point; (iv) measuring a second level ofcleaved HMWK in the second biological sample; and (v) assessingdevelopment of the autoimmune disease in the subject based on the changeof the levels of cleaved HMWK in the first and second biologicalsamples. If the second level of cleaved HMWK is higher than the firstlevel of cleaved HMWK, it indicates that the autoimmune diseaseprogresses in the subject or the subject has developed or is at risk fordeveloping the autoimmune disease.

In some embodiments, the first biological sample, the second biologicalsample, or both are serum samples or plasma samples. In otherembodiments, the first or second level of cleaved HMWK is measured by anassay that involves a binding agent (e.g., an antibody) specific tocleaved HMWK. In some examples, the assay is an enzyme-linkedimmunosorbent assay (ELISA) or an immunoblotting assay, e.g., aWesternblotting assay involving LiCor detection.

The method may further comprise subjecting the subject to a treatmentfor the autoimmune disease. In some embodiments, the subject isadministered with an effective amount of a plasma kallikrein (pKal)inhibitor such as those described herein.

Further, the present disclosure provides a method of assessing theefficacy of a treatment for an autoimmune disease (e.g., RA, CD, or UC)in a patient, the method comprising: (i) providing multiple biologicalsamples (e.g., serum samples or plasma samples) of a patient subjectedto a treatment for an autoimmune disease during the course of thetreatment; (ii) measuring the levels of high molecular weight kininogen(HMWK) in the multiple biological samples; and (iii) assessing theefficacy of the treatment in the patient based on the change of thelevels of cleaved HMWK over the course of the treatment. If the level ofcleaved HMWK decreases during the course of the treatment, it indicatesthat the treatment is effective in the patient.

In some embodiments, the treatment involves at least one plasmakallikrein inhibitor, e.g., those described herein. In otherembodiments, the levels of cleaved HMWK in the multiple biologicalsamples are measured by an assay that involves a binding agent (e.g., anantibody) specific to cleaved HMWK. In some examples, the assay is anenzyme-linked immunosorbent assay (ELISA) or an immunoblotting assay,e.g., a Western blotting assay involving LiCor detection. In any of themethods described herein, the biological sample used therein maycomprise a protease inhibitor or a protease inhibitor cocktail, which isadded to the biological sample after collection.

The kallikrein inhibitor useful in the methods may be, e.g., a plasmakallikrein (pKal) inhibitor. In some embodiments, the inhibitor is aplasma kallikrein inhibitor.

The kallikrein inhibitors useful in the methods may be any of the Kunitzdomain polypeptides known in the art or described herein, largerpolypeptides comprising any such Kunitz domains, provided the kallikreininhibitor polypeptides bind and inhibit kallikrein as determined instandard assays, kallikrein binding proteins (e.g., antibodies, e.g.,anti-plasma kallikrein antibodies), or other kallikrein inhibitorsdescribed herein.

Exemplary Kunitz domain peptides capable of inhibiting pKal activityincludes: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly ProCys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gin Cys GluGlu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gin Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2; DX-88), or a fragmentthereof, such as amino acids 3-60 of SEQ ID NO:2.

In some embodiments, the kallikrein inhibitor comprises or consists of aframework region of a kunitz domain and first and second binding loopregions of the DX-88 polypeptide.

In some embodiments, the kallikrein inhibitor comprises or consists ofan about 58-amino acid sequence of amino acids 3-60 of SEQ ID NO:2 orthe DX-88 polypeptide having the 60-amino acid sequence of SEQ ID NO:2.

In some embodiments, the kallikrein inhibitor comprises a plasmakallikrein binding protein (e.g., antibody, e.g., an anti-plasmakallikrein antibody described herein).

In some embodiments, the binding protein (e.g., antibody, e.g., humanantibody) binds the same epitope or competes for binding with a proteindescribed herein.

In some embodiments, the protein described herein is selected from thegroup consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01(also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-01, X115-F02, X124-G01 (also referred to herein asDX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. Such bindingproteins are described, e.g., in PCT Publication WO2012/094587 and USPatent Application Publication US 20100183625, both of which areincorporated herein by reference in their entirety.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X81-B01, X67-D03, X101-A01, M162-A04,X115-F02, X124-G01, or X63-G06.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein), but binds to the active formof plasma kallikrein (e.g., human plasma kallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasmakallikrein.

In some embodiments, the protein binds to one or more amino acids thatform the catalytic triad of plasma kallikrein: His434, Asp483, and/orSer578 (numbering based on the human sequence). In other embodiments,the protein binds to one or more amino acids of Ser479, Tyr563, and/orAsp585 (numbering based on the human sequence). In yet otherembodiments, the plasma kallikrein binding protein binds one or moreamino acids of: Arg 551, Gin 553, Tyr 555, and/or Arg 560 (amino acidposition numbering based on the human kallikrein sequence). In stillother embodiments, the plasma kallikrein binding protein binds one ormore amino acids of: Ser 478, Asn 481. Ser 525, and/or Lys 526 (aminoacid position numbering based on the human kallikrein sequence).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has anapparent inhibition constant (K_(i,app)) of less than 1000, 500, 100, or10 nM.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the plasmakallikrein binding protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.The plasma kallikrein binding protein can be a soluble Fab (sFab).

In other implementations the plasma kallikrein binding protein includesa Fab2′, scFv, minibody, scFv::Fc fusion, Fah::HSA fusion, HSA::Fahfusion, Fab::HSA::Fah fusion, or other molecule that comprises theantigen combining site of one of the binding proteins herein. The VH andVL regions of these Fabs can be provided as IgG Fab. Fab2, Fab2′, scFv.PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC,HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriateconstruction.

In one embodiment, the plasma kallikrein binding protein is a human orhumanized antibody or is non-immunogenic in a human. For example, theprotein includes one or more human antibody framework regions, e.g., allhuman framework regions.

In one embodiment, the plasma kallikrein binding protein includes ahuman Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99%identical to a human Fc domain.

In one embodiment, the plasma kallikrein binding protein is a primate orprimatized antibody or is non-immunogenic in a human. For example, theprotein includes one or more primate antibody framework regions, e.g.,all primate framework regions.

In one embodiment, the plasma kallikrein binding protein includes aprimate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or99% identical to a primate Fc domain. “Primate” includes humans (Homosapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)),gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes(Daubentonia madagascariensis), and tarsiers.

In one embodiment, the plasma kallikrein binding protein includes humanframework regions, or framework regions that are at least 95, 96, 97,98, or 99% identical to human framework regions.

In certain embodiments, the plasma kallikrein binding protein includesno sequences from mice or rabbits (e.g., is not a murine or rabbitantibody).

In some embodiments, the binding protein (e.g., antibody such as humanantibody) comprises a heavy chain immunoglobulin variable domainsequence and a light chain immunoglobulin variable domain sequence,wherein: the heavy chain immunoglobulin variable domain sequencecomprises one, two, or three (e.g., three) CDR regions from the heavychain variable domain of a protein described herein, and/or the lightchain immunoglobulin variable domain sequence comprises one, two, orthree (e.g., three) CDR regions from the light chain variable domain ofa protein described herein, wherein the protein binds to (e.g., andinhibits) plasma kallikrein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises one, two, or three (e.g., three) CDR regions from theheavy chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03,X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred toherein as DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04,and/or the light chain immunoglobulin variable domain sequence comprisesone, two, or three (e.g., three) CDR regions from the light chainvariable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (alsoreferred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930),X115-G04, M29-D09, M145-D11, M06-D09 and M35-G4304 (respectively).

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X81-B01 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X81-B01 or from X67-D03.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein asDX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04,X115-807, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X124-G01 (also referred to herein as DX-2930), X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04, and/or the light chain immunoglobulinvariable domain sequence comprises the light chain variable domain ofM162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to hereinas DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X124-001 (also referred to herein as DX-2930), X115-004, M29-D09,M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X81-B01, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X81-B01.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X67-D03, X101-A01,M162-A04, X115-F02, X124-G01, or X63-G06 and/or the light chainimmunoglobulin variable domain sequence comprises the light chainvariable domain of X67-D03, X101-A01, M162-A04, X115-F02, X124-G01, orX63-G06 (respectively).

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some embodiments, the protein comprises the heavy chain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein asDX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-1106, X115-A03, X115-D01, X115-F02,X124-G01 (also referred to herein as DX-2930), X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the protein comprises the heavy chain of X81-B01,and/or the light chain of X81-B01.

In some embodiments, the protein comprises the heavy chain of X67-D03,X101-A01, M162-A04, X115-F02, X124-G01, or X63-G06 and/or the lightchain of X67-D03, X101-A01, M162-A04, X115-F02, X124-G01, or X63-G06(respectively).

In some embodiments, the protein includes one or more of the followingcharacteristics: (a) a human CDR or human framework region; (b) the HCimmunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identical to a CDR of a HC variable domain describedherein; (c) the LC immunoglobulin variable domain sequence comprises oneor more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LCvariable domain described herein; (d) the LC immunoglobulin variabledomain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical to a LC variable domain described herein(e.g., overall or in framework regions or CDRs); (e) the HCimmunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variabledomain described herein (e.g., overall or in framework regions or CDRs);(f) the protein binds an epitope bound by a protein described herein, orcompetes for binding with a protein described herein; (g) a primate CDRor primate framework region; (h) the HC immunoglobulin variable domainsequence comprises a CDR1 that differs by at least one amino acid but byno more than 2 or 3 amino acids from the CDR1 of a HC variable domaindescribed herein; (i) the HC immunoglobulin variable domain sequencecomprises a CDR2 that differs by at least one amino acid but by no morethan 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variabledomain described herein; (j) the HC immunoglobulin variable domainsequence comprises a CDR3 that differs by at least one amino acid but byno more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variabledomain described herein; (k) the LC immunoglobulin variable domainsequence comprises a CDR1 that differs by at least one amino acid but byno more than 2, 3, 4, or 5 amino acids from the CDR1 of a LC variabledomain described herein; (1) the LC immunoglobulin variable domainsequence comprises a CDR2 that differs by at least one amino acid but byno more than 2, 3, or 4 amino acids from the CDR2 of a LC variabledomain described herein; (m) the LC immunoglobulin variable domainsequence comprises a CDR3 that differs by at least one amino acid but byno more than 2, 3, 4, or 5 amino acids from the CDR3 of a LC variabledomain described herein; (n) the LC immunoglobulin variable domainsequence differs by at least one amino acid but by no more than 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids from a LC variable domain describedherein (e.g., overall or in framework regions or CDRs); and (o) the HCimmunoglobulin variable domain sequence differs by at least one aminoacid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids froma HC variable domain described herein (e.g., overall or in frameworkregions or CDRs).

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, or 10 nM.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of antibodies selected fromthe group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01(also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein asDX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (alsoreferred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930),X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-1108, X63-06, X101-A01 (alsoreferred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-BO 1,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930),X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and/or heavy antibody variable regions of anantibody selected from the group consisting of M162-A04, M160-G12,M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922),X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07,X115-D05, X115-E09, X115-1106, X1115-A03, X115-D01, X115-F02, X124-G01(also referred to herein as DX-2930), X115-004, M29-D09, M145-D11,M06-D09 and M35-G04.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein), but binds to the active formof plasma kallikrein (e.g., human plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has anapparent inhibition constant (K_(i,app)) of less than 1000, 500, 100, or10 nM.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

The details of one or more embodiments of the present disclosure presentdisclosure are set forth in the accompanying drawings and thedescription below. Other features, objects, and advantages of thepresent disclosure will be apparent from the description and drawings,and from the claims.

The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing the association of cleaved HMWK withrheumatoid arthritis, Crohn's disease, and ulcerative colitis.

DETAILED DESCRIPTION

The present disclosure is based on the unexpected discovery thatelevated levels of cleaved HMWK were observed in patients having RA, CD,or UC. In particular, an extensive level of cleaved HMWK was observed inRA patients and a moderate level of cleaved HMWK was observed in both CDand UC patients. Accordingly, provided herein are new diagnostic andprognostic methods for identifying subjects having or at risk fordeveloping an autoimmune disease such as RA. UC, or CD, monitoringprogress of the autoimmune disease, and assessing the efficacy of atreatment for the autoimmune disease in a subject based on the level ofcleaved HMWK in a biological sample of the subject. Also describedherein are methods for treating such an autoimmune disease, as well asother diseases associated with the plasma kallikrein (pKal) system usinga pKal inhibitor such as those described herein.

Definitions

For convenience, before further description of the present disclosure,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “antibody” refers to a protein that includes at least oneimmunoglobulin variable domain or immunoglobulin variable domainsequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies. Fab and sFabfragments, F(ab′)₂, Fd fragments. Fv fragments, scFv, and domainantibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996;26(3):629-39)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). Antibodies may be from any source, but primate (human andnon-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see. Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk).Kabat definitions are used herein. Each VH and VL is typically composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. In IgGs, the heavy chainconstant region includes three immunoglobulin domains, CH1, CH2 and CH3.The light chain constant region includes a CL domain. The variableregion of the heavy and light chains contains a binding domain thatinteracts with an antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.The light chains of the immunoglobulin may be of types kappa or lambda.In one embodiment, the antibody is glycosylated. An antibody can befunctional for antibody-dependent cytotoxicity and/orcomplement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1 HC CDR2, HC CDR3, LC CDR1. LC CDR2, and LC CDR3. Each ofthe light chain CDRs can be human. HC CDR3 can be human. One or more ofthe framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of theHC or LC. For example, the Fc region can be human. In one embodiment,all the framework regions are human, e.g., have a sequence of aframework of an antibody produced by a human somatic cell, e.g., ahematopoietic cell that produces immunoglobulins or a non-hematopoieticcell. In one embodiment, the human sequences are germline sequences,e.g., encoded by a germline nucleic acid. In one embodiment, theframework (FR) residues of a selected Fab can be converted to theamino-acid type of the corresponding residue in the most similar primategermline gene, especially the human germline gene. One or more of theconstant regions can be human or effectively human. For example, atleast 70, 75, 80, 85, 90, 92, 95, 98, or 100% of an immunoglobulinvariable domain, the constant region, the constant domains (CH1, CH2,CH3, CL1), or the entire antibody can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and TgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the many immunoglobulinvariable region genes. Full-length immunoglobulin “light chains” (about25 KDa or about 214 amino acids) are encoded by a variable region geneat the NH2-terminus (about 110 amino acids) and a kappa or lambdaconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 KDa or about 446 amino acids), are similarlyencoded by a variable region gene (about 116 amino acids) and one of theother aforementioned constant region genes, e.g., gamma (encoding about330 amino acids). The length of human HC varies considerably because HCCDR3 varies from about 3 amino-acid residues to over 35 amino-acidresidues.

The term “antigen-binding fragment” of a full length antibody refers toone or more fragments of a full-length antibody that retain the abilityto specifically bind to a target of interest. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of afull length antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, abivalent fragment including two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andCH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains functionality.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos.5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883.

Antibody fragments can be obtained using any appropriate techniqueincluding conventional techniques known to those with skill in the art.The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition.” which as used herein refer to a preparation ofantibodies or fragments thereof of single molecular composition,irrespective of how the antibody was generated.

The inhibition constant (Ki) provides a measure of inhibitor potency; itis the concentration of inhibitor required to reduce enzyme activity byhalf and is not dependent on enzyme or substrate concentrations. Theapparent Ki (K_(i,app)) is obtained at different substrateconcentrations by measuring the inhibitory effect of differentconcentrations of inhibitor (e.g., inhibitory binding protein) on theextent of the reaction (e.g., enzyme activity); fitting the change inpseudo-first order rate constant as a function of inhibitorconcentration to the Morrison equation (Equation 1) yields an estimateof the apparent Ki value. The Ki is obtained from the y-interceptextracted from a linear regression analysis of a plot of Ki,app versussubstrate concentration.

$\begin{matrix}{v = {v_{o} - {v_{o}\left( \frac{\left( {K_{i,{app}} + I + E} \right) - \sqrt{\left( {K_{i,{app}} + I + E} \right)^{2} - {4 \cdot I \cdot E}}}{2 \cdot E} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where v=measured velocity; v₀=velocity in the absence of inhibitor;K_(i,app)=apparent inhibition constant; I=total inhibitor concentration;and E=total enzyme concentration.

As used herein. “binding affinity” refers to the apparent associationconstant or K_(a). The K_(a) is the reciprocal of the dissociationconstant (K_(d)). A binding protein may, for example, have a bindingaffinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M⁻¹ for aparticular target molecule. Higher affinity binding of a binding proteinto a first target relative to a second target can be indicated by ahigher K_(a) (or a smaller numerical value K_(d)) for binding the firsttarget than the K_(a) (or numerical value K_(d)) for binding the secondtarget. In such cases, the binding protein has specificity for the firsttarget (e.g., a protein in a first conformation or mimic thereof)relative to the second target (e.g., the same protein in a secondconformation or mimic thereof; or a second protein). Differences inbinding affinity (e.g., for specificity or other comparisons) can be atleast 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000,or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inTRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂ at pH7.5). Thesetechniques can be used to measure the concentration of bound and freebinding protein as a function of binding protein (or target)concentration. The concentration of bound binding protein ([Bound]) isrelated to the concentration of free binding protein ([Free]) and theconcentration of binding sites for the binding protein on the targetwhere (N) is the number of binding sites per target molecule by thefollowing equation:[Bound]=N·[Free]/((1/K _(a))+[Free]).

It is not always necessary to make an exact determination of K_(a),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(a), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

The term “binding protein” refers to a protein that can interact with atarget molecule. This term is used interchangeably with “ligand.” A“plasma kallikrein binding protein” refers to a protein that caninteract with (e.g., bind) plasma kallikrein, and includes, inparticular, proteins that preferentially or specifically interact withand/or inhibit plasma kallikrein. A protein inhibits plasma kallikreinif it causes a decrease in the activity of plasma kallikrein as comparedto the activity of plasma kallikrein in the absence of the protein andunder the same conditions. In some embodiments, the plasma kallikreinbinding protein is an antibody.

The term “kallikrein inhibitor” refers to any agent or molecule thatinhibits kallikrein.

The term “combination” refers to the use of the two or more agents ortherapies to treat the same patient, wherein the use or action of theagents or therapies overlap in time. The agents or therapies can beadministered at the same time (e.g., as a single formulation that isadministered to a patient or as two separate formulations administeredconcurrently) or sequentially in any order.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

It is possible for one or more framework and/or CDR amino acid residues(or binding loop amino acid residues) of a binding protein to includeone or more mutations (e.g., substitutions (e.g., conservativesubstitutions or substitutions of non-essential amino acids),insertions, or deletions) relative to a binding protein describedherein. A plasma kallikrein binding protein may have mutations (e.g.,substitutions (e.g., conservative substitutions or substitutions ofnon-essential amino acids), insertions, or deletions) (e.g., at leastone, two, three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4,3, or 2 mutations) relative to a binding protein described herein, e.g.,mutations which do not have a substantial effect on protein function.The mutations can be present in framework regions, CDRs (or bindingloops), and/or constant regions. In some embodiments, the mutations arepresent in a framework region. In some embodiments, the mutations arepresent in a CDR. In some embodiments, the mutations are present in aconstant region. Whether or not a particular substitution will betolerated, i.e., will not adversely affect biological properties, suchas binding activity can be predicted, e.g., by evaluating whether themutation is conservative or by the method of Bowie, et al. (1990)Science 247:1306-1310.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

An “epitope” refers to the site on a target compound that is bound by abinding protein (e.g., an antibody such as a Fab or full lengthantibody). In the case where the target compound is a protein, the sitecan be entirely composed of amino acid components, entirely composed ofchemical modifications of amino acids of the protein (e.g., glycosylmoieties), or composed of combinations thereof. Overlapping epitopesinclude at least one common amino acid residue, glycosyl group,phosphate group, sulfate group, or other molecular feature.

A first binding protein (e.g., antibody) “binds to the same epitope” asa second binding protein (e.g., antibody) if the first binding proteinbinds to the same site on a target compound that the second bindingprotein binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%,80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence orother molecular feature (e.g., glycosyl group, phosphate group, orsulfate group)) with the site that the second binding protein binds.

A first binding protein (e.g., antibody) “competes for binding” with asecond binding protein (e.g., antibody) if the binding of the firstbinding protein to its epitope decreases (e.g., by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second bindingprotein that binds to its epitope. The competition can be direct (e.g.,the first binding protein binds to an epitope that is the same as, oroverlaps with, the epitope bound by the second binding protein), orindirect (e.g., the binding of the first binding protein to its epitopecauses a steric change in the target compound that decreases the abilityof the second binding protein to bind to its epitope).

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% ofthe length of the reference sequence. For example, the referencesequence may be the length of the immunoglobulin variable domainsequence.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified to include a sufficient number of humanframework amino acid positions such that the immunoglobulin variableregion does not elicit an immunogenic response in a normal human.Descriptions of “humanized” immunoglobulins include, for example, U.S.Pat. Nos. 6,407,213 and 5,693,762.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons. N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either can beused. Specific hybridization conditions referred to herein are asfollows: (1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); (2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) highstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C. followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions and the onesthat should be used unless otherwise specified. The disclosure includesnucleic acids that hybridize with low, medium, high, or very highstringency to a nucleic acid described herein or to a complementthereof, e.g., nucleic acids encoding a binding protein describedherein. The nucleic acids can be the same length or within 30, 20, or10% of the length of the reference nucleic acid. The nucleic acid cancorrespond to a region encoding an immunoglobulin variable domainsequence described herein.

An “isolated composition” refers to a composition that is removed fromat least 90% of at least one component of a natural sample from whichthe isolated composition can be obtained. Compositions producedartificially or naturally can be “compositions of at least” a certaindegree of purity if the species or population of species of interests isat least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on aweight-weight basis.

An “isolated” protein refers to a protein that is removed from at least90% of at least one component of a natural sample from which theisolated protein can be obtained. Proteins can be “of at least” acertain degree of purity if the species or population of species ofinterest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pureon a weight-weight basis.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas changing an “essential” amino acid residueresults in a substantial loss of activity.

A “patient”. “subject” or “host” (these terms are used interchangeably)to be treated by the method may mean either a human or non-human animal.

A “subject in need thereof” includes, for example, a subject having adisease or disorder described herein or a subject at risk for developinga disease or disorder described herein.

The term “kallikrein” (e.g., plasma kallikrein) refers to peptidases(enzymes that cleave peptide bonds in proteins), a subgroup of theserine protease family. Plasma kallikrein cleaves kininogen to generatekinins, potent pro-inflammatory peptides. DX-88 (also referred to hereinas “PEP-1”) is a potent (Ki<1 nM) and specific inhibitor of plasmakallikrein (NP_000883). (See also e.g., WO 95/21601 or WO 2003/103475).

The amino acid sequence of KLKb1 (plasma kallikrein) is:

KLKb1 >gi|78191798|ref|NP_000883.2| plasma kallikreinB1 precursor [Homo sapiens] (SEQ ID NO: 3)      MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRIGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISCYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQEICTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDIPFSOIKEIIIHQNYKVSEGNHDIALIKLQAPLNYIEFQKPICLPSKGDISIIYINCWVIGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA.

As used herein the term “DX-2922” as used interchangeably with the term“X101-A01”. Other variants of this antibody are described below.

Antibody Identification Description X63-G06 Non-germlined Fab discoveredusing ROLIC, same HC but different LC as M160-G12 X81-B01 Germlined IgGproduced in HEK 293T cells X101-A01 Germlined IgG produced in CHO cells,same HC and LC sequence as X81-B01 DX-2922 Alternate nomenclature forX101-A01

As used herein the term “DX-2930” as used interchangeably with the term“X124-G01”. Other variants of this antibody are described below.

Antibody Identification Description M162-A04 Non-germlined Fabdiscovered using phage display M199-A08 Heavy chain CDR3 varied Fabderived by affinity maturation of M162-A04 X115-F02 Germlined Fabproduced in 293T cells, same variable heavy chain as X124-G01 X124-001or Germlined IgG produced in CHO cells, LC and HC DX-2930 sequence asX115-F02 except that the C-terminal Lys of the HC is removed in X124-G01 (also known as DX-2930).KLK1

>gi|13529059|gb|AAH05313.1| Kallikrein 1 [Homo sapiens] (SEQ ID NO: 4)MWFLVLCLALSLGGTGAAPPIQSRIVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTAAHCISDNYQLWLGRHNLFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDLMLLRLTEPADTITDAVKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECKKVHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSY VKWIEDTIAENS

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term “preventing” a disease in a subject refers to subjecting thesubject to a pharmaceutical treatment, e.g., the administration of adrug, such that at least one symptom of the disease is prevented, thatis, administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal) sothat it protects the host against developing the unwanted condition.“Preventing” a disease may also be referred to as “prophylaxis” or“prophylactic treatment.”

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, because a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount is likely but not necessarily lessthan the therapeutically effective amount.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleicacid sequence that contains a sufficient number of identical orequivalent (e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleic acid sequence such that the first and second amino acid ornucleic acid sequences have (or encode proteins having) similaractivities, e.g., a binding activity, a binding preference, or abiological activity. In the case of antibodies, the second antibody hasthe same specificity and has at least 50%, at least 25%, or at least 10%of the affinity relative to the same antigen.

Sequences similar or homologous (e.g., at least about 85% sequenceidentity) to the sequences disclosed herein are also part of thisapplication. In some embodiments, the sequence identity can be about85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In someembodiments, a plasma kallikrein binding protein can have about 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequenceidentity to a binding protein described herein. In some embodiments, aplasma kallikrein binding protein can have about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the HCand/or LC framework regions (e.g., HC and/or LC FR 1, 2, 3, and/or 4) toa binding protein described herein. In some embodiments, a plasmakallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LCCDRs (e.g., HC and/or LC CDR1, 2, and/or 3) to a binding proteindescribed herein. In some embodiments, a plasma kallikrein bindingprotein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher sequence identity in the constant region (e.g., CH1, CH2,CH3, and/or CL1) to a binding protein described herein.

In addition, substantial identity exists when the nucleic acid segmentshybridize under selective hybridization conditions (e.g., highlystringent hybridization conditions), to the complement of the strand.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form.

Motif sequences for biopolymers can include positions which can bevaried amino acids. For example, the symbol “X” in such a contextgenerally refers to any amino acid (e.g., any of the twenty naturalamino acids) unless otherwise specified, e.g., to refer to anynon-cysteine amino acid. Other allowed amino acids can also be indicatedfor example, using parentheses and slashes. For example, “(A/W/F/N/Q)”means that alanine, tryptophan, phenylalanine, asparagine, and glutamineare allowed at that particular position.

Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02. Particular binding proteins may show a difference, e.g., inspecificity or binding, that are statistically significant (e.g., Pvalue<0.05 or 0.02). The terms “induce”, “inhibit”, “potentiate”,“elevate”, “increase”, “decrease” or the like, e.g., which denotedistinguishable qualitative or quantitative differences between twostates, and may refer to a difference, e.g., a statistically significantdifference, between the two states.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the proteinto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effect ofthe composition is outweighed by the therapeutically beneficial effects.

A “therapeutically effective dosage” preferably modulates a measurableparameter of a disease or disorder. For example, a therapeuticallyeffective dosage can reduce the degree of a symptom of the disease ordisorder by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% as compared to the symptom prior to treatment. Theability of a compound to modulate a measurable parameter, e.g., adisease-associated parameter, can be evaluated in an animal model systempredictive of efficacy in human disorders and conditions. Alternatively,this property of a composition can be evaluated by examining the abilityof the compound to modulate a parameter in vitro.

“Treating” a disease or disorder in a subject (including, e.g.,“treating” a subject having or at risk for developing a disease ordisorder) refers to subjecting the subject to a pharmaceuticaltreatment, e.g., the administration of a drug, such that at least onesymptom of the disease is prevented, cured, alleviated decreased, or thelike.

A “disease associated with protein misfolding or aggregation” is adisease that arises, at least in part, due to a change (e.g., anobstruction) in the folding process or the stability of the foldedstructure of a protein. Obstruction of the folding process, increases inaggregation and de-stabilizing the native protein structure may causeloss-of-function or gain-of-function pathologies. A disease associatedwith protein misfolding or aggregation includes, but is not limited to:systemic amyloidosis, cryoglobulinemia, and sickle cell disease.Neurological diseases associated with protein misfolding or aggregationinclude Jacob-Kreutzfeld disease (associated with prion proteins),Alzheimer's disease, other amyloid diseases such as Familial amyloidoticpolyneuropathy (FAP).

I. Use of Cleaved High Molecule Weight Kininogen (HMWK) as a Biomarkerin Diagnosis and Prognosis Assays for Autoimmune Diseases

Unexpectedly, elevated levels of cleaved HMWK were found in autoimmunediseases such as Rheumatoid Arthritis (RA), Crohn's Disease (CD) andUlcerative Colitis (UC). Example 1 below. Thus, cleaved HMWK can serveas a reliable biomarker for diagnosing an autoimmune disease (e.g., RA,UC, and CD), monitoring the progress of such an autoimmune disease, andassessing the efficacy of a treatment for the disease.

Accordingly, described herein are diagnostic and prognostic methods foran autoimmune disease (e.g., RA, UC, and CD) based on the level ofcleaved HMWK in a biosample (e.g., a plasma sample) obtained from acandidate patient.

High-molecular-weight kininogen (HMWK), also known as theWilliams-Fitzgerald-Flaujeac factor or the Fitzgerald factor or theHMWK-kallikrein factor, is a protein from the blood coagulation systemas well as the kinin-kallikrein system. It is a protein that adsorbs tothe surface of biomaterials that come in contact with blood in vivo.High molecular-weight kininogen (HMWK) exists in the plasma as a singlepolypeptide (1-chain) multi-domain (domains 1-6) protein with amolecular weight of approximately 110 kDa. HMWK is cleaved by pKalwithin domain 4 to release the 9 amino acid, pro-inflammatory peptidebradykinin and a 2-chain form of HMWK (cleaved kininogen). The 2 chainsof HMWK are the heavy chain, which contains the domains 1-3 of HMWK, andthe light chain, which contains the domains 5 and 6 of HMWK. The heavyand light chains have a molecular weight of approximately 56 and 46kiloDaltons, respectively.

The human gene encoding HMWK is kininogen 1 (KNG1), KNG1 is transcribedand alternatively spliced to form mRNAs that encode either HMWK or lowmolecular weight kininogen (LMWK). An exemplary protein sequence of HMWKis provided below:

>gi|156231037|ref|NP_001095886.1| kininogen-1isoform 1 precursor [Homo sapiens] (SEQ ID NO: 5)MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAAIGECIATVGKRSSTKFSVATQTCQITPAEGPVVIAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKAPVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGEGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

Intact high molecular weight kininogen (HMWK) can be assayed, forexample, using coagulant or immunological methods, e.g.,radioimmunoassay (see, e.g., Kerbiriou-Nabias, D. M., Br J Haematol,1984, 56(2):2734-86). A monoclonal antibody to the light chain of humanHMWK is known. See, e.g., Reddigari, S. R. & Kaplan, A. P., Blood, 1999,74:695-702. An assay for HMWK that relies on a chromogenic substrate canalso be used. See, e.g., Scott. C. F. et al. Thromb Res, 1987,48(6):685-700; Gallimore, M. J. et al. Thromb Res. 2004, 114(2):91-96.

Cleaved high molecular weight kininogen (HMWK), also referred to hereinas “cleaved kininogen,” can be assessed, for example, using methodsdescribed in Example 1, e.g., Western blot. Antibodies that specificallybind cleaved HMWK, such as, e.g., the mouse mAb clone 11H05 can be used.Additionally, cleaved HMWK may be assessed using mass spectrometry.Immunoblotting techniques for assessing levels of cleaved HMWK are knownin the art. See, e.g., Buhler R. et al. Blood Coagul Fibrinolysis, 1995,6(3):223-232.

Exemplary sequences of the heavy and light chains of cleaved kininogenare provided below.

>cleaved kininogen-1 heavy chain (SEQ ID NO: 6)QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRIIEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTAIVGKRSSTKFSVATQTCQITPAEGPVVTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKYIYPTVNCQPLGMISLMK >cleaved kininogen-1 light chain (SEQ ID NO: 7)SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDL TDGLS

In some examples, the levels of intact HMWK and cleaved HMWK aremeasured by a Western blot analysis, e.g., a Simple Western™ ProteinSimple® Western blot analysis. Simple Western™ assays are known in theart (see, e.g., Rustandi et al. Qualitative and quantitative evaluationof Simon™, a new CE-based automated Western blot system as applied tovaccine development. Electrophoresis. 2012 September; 33(17):2790-7).Simple Western™ products are also available commercially (see, e.g.,ProteinSimple®, Santa Clara, Calif.).

To practice any of the diagnostic and/or prognostic methods describedherein, a biosample (e.g., a biofluid sample such as a plasma sample ora serum sample) can be obtained from a candidate subject (e.g., acandidate human patient) for measuring the level of cleaved HMWK. Asubject can be a mammal, more preferably a human. Non-human mammalsinclude, but are not limited to, farm animals, sport animals, pets,primates, horses, dogs, cats, mice and rats. A human subject may be ahuman patient suspected of having an autoimmune disease such as thosedescribed herein, e.g., RA, CD, or UC.

The biosample obtained from the subject may be a tissue or fluid sample.Examples of fluid samples include, but are not limited to, saliva,blood, plasma, serum, and urine. In some embodiments, the biosample fromthe subject comprises leukocytes, e.g., a blood sample. The biosamplemay be obtained from the patient using any method known in the art,e.g., venipuncture, biopsy, or swab. Prior to analysis, a proteaseinhibitor or a protease inhibitor cocktail may be added to the biosampleto inhibit cleavage of HMWK in vitro. Any protease inhibitor known inthe art can be used in the methods described herein.

The level of cleaved HMWK can be measured by any suitable assay known inthe art (see, e.g., Molecular Cloning: A Laboratory Manual. J. Sambrook,et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001, Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Microarraytechnology is described in Microarray Methods and Protocols, R. Matson,CRC Press, 2009, or Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons. Inc., New York).

In some embodiments, the level of the cleaved HMWK protein in the sampleis measured. Assays for detecting cleaved HMWK protein levels include,but are not limited to, immunoassays (also referred to herein asimmune-based or immuno-based assays, e.g., Western blot,immunohistochemistry and ELISA assays), Mass spectrometry, and multiplexbead-based assays. Such assays for protein level detection are known inthe art.

In some examples, the level of cleaved HMWK is measured by a Westernblot assay, which may involve LiCor detection as described herein. Inother examples, the level of cleaved HMWK protein is measured by animmunohistochemistry assay, which may involve a binding partner, such asan antibody, that specifically binds the cleaved HMWK or specificallybinds to cleaved and uncleaved HMWK.

Binding partners for protein detection can be designed using methodsknown in the art and as described herein. In some embodiments, thecleaved HMWK protein binding partners, e.g., anti-cleaved HMWKantibodies, bind to a part of or an entire amino acid sequence of theHMWK protein. Other examples of protein detection and quantitationmethods include multiplexed immunoassays as described for example inU.S. Pat. Nos. 6,939,720 and 8,148,171, and published US PatentApplication No. 2008/0255766, and protein microarrays as described forexample in published US Patent Application No. 2009/0088329. Anysuitable binding partner for cleaved HMWK is contemplated for detectionof a cleaved HMWK level. In some embodiments, the binding partner is anymolecule that binds specifically to a HMWK protein or a cleaved HMWKprotein. Such a binding partner may bind to the cleaved version of HMWK(e.g., the cleaved HMWK) with much higher affinity as compared to itsbinding to the uncleaved HMWK. In some instances, the binding partnersuch as an antibody may bind only to the cleaved version of HMWK.

The antibody to be used in the method described herein can be in anyform, including, but not limited to, a full-length antibody or anantigen-binding fragments thereof, such as Fab, F(ab)2. Fv, single chainantibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, scFv, or dAbfragments. Methods for producing antibodies are well known in the art(see, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual”(2nd Ed.), Cold Spring Harbor Laboratory Press (1989): Lewin, “GenesIV”, Oxford University Press, New York, (1990), and Roitt et al.,“Immunology” (2nd Ed.), Gower Medical Publishing, London, N.Y. (1989).WO2006/040153, WO2006/122786, and WO2003/002609). See also descriptionsherein.

In other embodiments, the binding partners used for measuring the levelof cleaved HMWK can be non-antibody peptide molecules or aptamers thatbind specifically to cleaved HMWK. Methods for producing peptidemolecules and aptamers are also known in the art (see, e.g., publishedUS Patent Application No. 2009/0075834, U.S. Pat. Nos. 7,435,542,7,807,351, and 7,239,742).

Once the level of the cleaved HMWK in a biosample obtained from acandidate subject is determined, it can be compared with a control levelfor determining whether the subject has, is at risk of, or suspected ofhaving an autoimmune disease, such as RA. CD, or UC.

In some embodiments, the control level is a level of cleaved HMWK in acontrol sample, such as a cell, tissue or fluid obtained from a healthysubject or population of healthy subjects, which preferably are of thesame species as the candidate subject. As used herein, a healthy subjectis a subject that is apparently free of the target disease (e.g., RA,CD, or UC) at the time the HMWK level is measured or has no history ofthe disease.

In some embodiments, a control level is a level of cleaved HMWK that isundetectable or below a background/noise level obtained using a standardmethod of detection (e.g., Western blot or immunohistochemistry).Preferably, the standard method of detection is the same method used formeasuring the level of cleaved HMWK in the sample of the candidatesubject.

The control level can also be a predetermined level. Such apredetermined level can represent the level of the cleaved HMWK in apopulation of subjects that do not have or are not at risk for anautoimmune disease as described herein. The predetermined level can takea variety of forms. For example, it can be single cut-off value, such asa median or mean. In some embodiments, such a predetermined level can beestablished based upon comparative groups, such as where one definedgroup is known to have a target autoimmune disease (e.g., RA, UC, or CD)and another defined group is known to not have the target autoimmunedisease. Alternatively, the predetermined level can be a range, forexample, a range representing the levels of the cleaved HWMK in acontrol population within a predetermined percentile.

The predetermined level can depend upon the particular populationselected. For example, an apparently healthy (no detectable disease orprior history of a target autoimmune disease, such as RA, CD, or UC)will have a different ‘normal’ range of cleaved HMWK than will apopulation the members of which have or is at risk for the targetautoimmune disease, which may be in remission. Accordingly, thepredetermined levels selected may take into account the category inwhich a subject falls. Appropriate ranges and categories can be selectedwith no more than routine experimentation by those of ordinary skill inthe art.

The control level as described herein can be determined by routinetechnology. In some examples, the control level can be obtained byperforming a conventional method (e.g., the same assay for obtaining thelevel of cleaved HMWK in a test sample as described herein) on a controlsample as also described herein. In other examples, levels of cleavedHMWK can be obtained from members of a control population and theresults can be analyzed by, e.g., a computational program, to obtain thecontrol level (a predetermined level) that represents the level ofcleaved HWMK in the control population.

By comparing the level of cleaved HMWK of a sample obtained from acandidate subject to the control level as described herein, it can bedetermined as to whether the candidate subject has or is at risk for atarget autoimmune disease. For example, if the level of the cleaved HMWKof the candidate subject deviates from the control level (e.g., elevatedor decreased as compared to the control level), the candidate subjectmight be identified as having or at risk for the target autoimmunedisease.

As used herein. “an elevated level or a level above a control” meansthat the level of cleaved HMWK is higher than a control level, such as apre-determined threshold or a level of cleaved HMWK in a control sample.Control levels are described in detail herein. An elevated level ofcleaved HMWK includes a cleaved HMWK level that is, for example, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%,400%, 500% or more above a control level. An elevated level of cleavedHMWK also includes increasing a phenomenon from a zero state (e.g., noor undetectable cleaved HMWK in a control) to a non-zero state (e.g.,some cleaved HMWK or detectable cleaved HMWK in a sample).

As used herein, “a decreased level or a level below a control” meansthat the level of cleaved HMWK is lower than a control level, such as apre-determined threshold or a level of cleaved HMWK in a control sample.Control levels are described in detail herein. An decreased level ofcleaved HMWK includes a cleaved HMWK level that is, for example, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%,400%, 500% or more lower than a control level. A decreased level ofcleaved HMWK also includes decreasing a phenomenon from a non-zero state(e.g., some cleaved HMWK or detectable cleaved HMWK in a sample) to azero state (e.g., no or undetectable cleaved HMWK in a control).

In some embodiments, if an extensive level (e.g., at least 40%, 50%,60%, 70%, 80%, 90%, or 100%) of cleaved HMWK is observed in a biosampleobtained from a RA candidate patient, that candidate is diagnosed ashaving or at risk for RA flare. If an moderate level (e.g., about10-30%) of cleaved HMWK is observed in a biosample obtained from a UC orCD candidate patient, that candidate is diagnosed as having or at riskfor UC or CD.

Further, the level of cleaved HMWK can be used as a biomarker formonitoring the development of an autoimmune disease, such as RA, UC, andCD. For example, at least two biosamples (e.g., serum samples or plasmasamples) can be obtained from a human subject having or at risk fordeveloping a target autoimmune disease such as RA, UC, or CD atdifferent time points. In some examples, the second biosample can beobtained at least 1 month (e.g., 3 months, 6 months, 9 months, or 12months) after the first biosample is obtained. The levels of cleavedHMWK can be measured in the at least two biosamples. If the level ofcleaved HMWK is elevated over time (e.g., the level of cleaved HMWK in alater obtained biosample is higher than that in an earlier obtainedbiosample by, e.g., at least 20%, 50%, 70%, 90%, 1-fold, 5-fold,10-fold, 20-fold, 50-fold, or 100-fold), it indicated disease progressin the subject (e.g., having a higher risk for developing the autoimmunedisease or the autoimmune disease exacerbates in the subject).

Moreover, the level of cleaved HMWK can also be used as a biomarker toassess the responsiveness of a subject to an anti-autoimmune treatment,e.g., those described herein. For example, multiple biosamples can beobtained from a human patient subjected to a treatment during the courseof the treatment and the levels of cleaved HMWK can be measuredfollowing routine technology such as those described herein. If thelevel of cleaved HMWK in a human patient subject to a treatment remainsdecreases over the course of the treatment (e.g., the level of cleavedHMWK in a later obtained biosample is lower than that in an earlierobtained biosample, e.g., by at least 20%, 50%, 70%, 80%, 90%, 100%,2-fold, 5-fold, 10-fold, 50-fold, or 100-fold), it indicates that thehuman patient is responsive to the treatment. On the other hand, if thelevel of cleaved HMWK remains substantially the same over the course ofthe treatment (e.g., the level of cleaved HMWK in a later obtainedbiosample is substantially identical to or decreases by less than 20%,e.g., 15%, 10%, or 5% relative to that of an earlier obtainedbiosample), it indicates that the human patient is not responsive to thetreatment.

When a subject is identified as having or at risk for an autoimmunedisease (e.g., RA, UC, or CD) by any of the methods described herein, asuitable treatment can be performed to treat the disease. In someexamples, the subject can be treated by one or more pKal inhibitors asdescribed herein. When a subject is determined as not responsive to atreatment by any of the methods described herein, a higher dose and/orfrequency of dosage of a therapeutic (e.g., a pKal inhibitor) can beadministered to the subject. Alternatively, the subject can switch to adifferent treatment. On the other hand, the dosage or frequency ofdosage of the therapeutic agent is maintained, lowered, or ceased in asubject identified as responsive to the treatment or not in need offurther treatment.

II. Treatment of Autoimmune Diseases

Also described herein are methods for treating diseases associated withthe plasma kallikrein (pKal) system, including, but not limited to,diabetic macular edema, retinal proliferation, brain trauma, acutespinal cord injury, localized amyloidosis, autoimmune diseases such aspsoriasis, multiple aclerosis, inflammatory bowel disease, rheumatoidarthritis, vasculitis, systemic lupus erythematosis nephritis, systemicmastocytosis, severe burns, and neuropathic pain (diabetic andpost-herpetic neuralgia). Such methods comprise administering to asubject in need of the treatment (e.g., a human patient having or atrisk for the disease) an effective amount of one or more kallikreininhibitors via a suitable route.

(A) Plasma Kallikrein

Exemplary plasma kallikrein sequences against which plasma kallikreinbinding proteins may be developed can include human, mouse, or ratplasma kallikrein amino acid sequences, a sequence that is 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, ora fragment thereof, e.g., of a sequence provided below.

The sequence of human plasma kallikrein that was used in selections andsubsequent screening of binding proteins is shown below (accessionnumber NP_000883.2). The human plasma kallikrein (86 kDa) that was usedwas purified from human plasma and activated with factor XIIa by acommercial vendor. Factor XIIa activates prekallikrein by cleaving thepolypeptide sequence at a single site (between Arg371-Ile372, cleavagesite marked by “/” in the sequence below) to generate active plasmakallikrein, which then consists of two disulfide linked polypeptides; aheavy chain of approximately 52 kDa and a catalytic domain ofapproximately 34 kDa [Colman and Schmaier, (1997) “Contact System: AVascular Biology Modulator With Anticoagulant, Profibrinolytic,Antiadhesive, and Proinflammatory Attributes” Blood, 90, 3819-3843]

The human, mouse, and rat prekallikrein amino acid sequences, and themRNA sequences encoding the same, are illustrated below. The sequencesof prekallikrein are the same as plasma kallikrein, except that activeplasma kallikrein (pkal) has the single polypeptide chain cleaved at asingle position (indicated by the “/”) to generate two chains. Thesequences provided below are full sequences that include signalsequences. On secretion from the expressing cell, it is expected thatthe signal sequences are removed.

Exemplary plasma kallikrein proteins from various species can be foundin GeneBank under accession numbers NP_000883.2 (human pKal protein),NM_000892 (human pKal mRNA), NP_032481.1 (mouse pKal protein),NM_008455.2 (mouse pKal mRNA). NP_036857.2 (rat pKal protein), andNM_012725 (rat pKal mRNA).

(B) Kallikrein Inhibitors

Kunitz Domain Inhibitors.

A number of useful inhibitors of kallikrein, either tissue and/or plasmakallikrein, include a Kunitz domain. Exemplary Kunitz domain inhibitorsare described in US Patent Application Publication US20100183625, whichis incorporated by reference herein.

As used herein, a “Kunitz domain” is a polypeptide domain having atleast 51 amino acids and containing at least two, and preferably three,disulfides. The domain is folded such that the first and sixthcysteines, the second and fourth, and the third and fifth cysteines formdisulfide bonds (e.g., in a Kunitz domain having 58 amino acids,cysteines can be present at positions corresponding to amino acids 5,14, 30, 38, 51, and 55, according to the number of the BPTI homologoussequences provided below, and disulfides can form between the cysteinesat position 5 and 55, 14 and 38, and 30 and 51), or, if two disulfidesare present, they can form between a corresponding subset of cysteinesthereof. The spacing between respective cysteines can be within 7, 5, 4,3, 2, 1 or 0 amino acids of the following spacing between positionscorresponding to: 5 to 55, 14 to 38, and 30 to 51, according to thenumbering of the BPTI sequence provided below. The BPTI sequence can beused as a reference to refer to specific positions in any generic Kunitzdomain. Comparison of a Kunitz domain of interest to BPTI can beperformed by identifying the best fit alignment in which the number ofaligned cysteines in maximized.

The 3D structure (at high resolution) of the Kunitz domain of BPTI isknown. One of the X-ray structures is deposited in the BrookhavenProtein Data Bank as “6PTI”. The 3D structure of some BPTI homologues(Eigenbrot et al., (1990) Protein Engineering, 3(7):591-598; Hynes etal., (1990) Biochemistry, 29:10018-10022) are known. At least eighty oneKunitz domain sequences are known. Known human homologues include threeKunitz domains of LACI also known as tissue factor pathway inhibitor(TFPI) (Wun et al., (1988) J. Biol. Chem. 263(13):6001-6004; Girard etal., (1989) Nature, 338:518-20; Novotny el al. (1989) J. Biol. Chem.,264(31):18832-18837) two Kunitz domains of Inter-α-Trypsin Inhibitor.APP-I (Kido et al., (1988) J. Biol. Chem., 263(34):18104-18107), aKunitz domain from collagen, three Kunitz domains of TFPI-2 (Sprecher etal., (1994) PNAS USA, 91:3353-3357), the Kunitz domains of hepatocytegrowth factor activator inhibitor type 1, the Kunitz domains ofHepatocyte growth factor activator inhibitor type 2, the Kunitz domainsdescribed in U.S. Patent Publication No.: 2004-0152633. LACI is a humanserum phosphoglycoprotein with a molecular weight of 39 kDa containingthree Kunitz domains.

The Kunitz domains above are referred to as LACI-K1 (residues 50 to107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The cDNAsequence of LACI is reported in Wun et al. (J. Biol. Chem., 1988,263(13):6001-6004). Girard et al. (Nature, 1989, 338:518-20) reportsmutational studies in which the P1 residues of each of the three Kunitzdomains were altered. LACI-K1 inhibits Factor Vila (F.VIIa) when F.VIIais complexed to tissue factor and LACI-K2 inhibits Factor Xa.

Proteins containing exemplary Kunitz domains include the following, withSWISS-PROT Accession Numbers in parentheses:

A4_HUMAN (P05067), A4_MACFA (P53601), A4_MACMU (P29216), A4_MOUSE(P12023), A4_RAT (P08592), A4_SAISC (Q95241), AMBP_PLEPL (P36992),APP2_HUMAN (Q06481), APP2_RAT (P15943), AXP1_ANTAF (P81547), AXP2_ANTAF(P81548), BPT1_BOVIN (P00974), BPT2_BOVIN (P04815), CA17_HUMAN (Q02388),CA36_CHICK (P15989), CA36_HUMAN (P12111), CRPI_BOOMI (P81162),ELAC_MACEU (O62845), ELAC_TRIVU (Q29143), EPPI_HUMAN (O95925),EPPI_MOUSE (Q9DA01), HTIB_MANSE (P26227), IBP_CARCR (P00993), IBPC_BOVIN(P00976), IBPI_TACTR (P16044), IBPS_BOVIN (P00975), ICS3_BOMMO (P07481),IMAP_DROFU (P11424), IP52_ANESU (P10280), ISC1_BOMMO (P10831),ISC2_BOMMO (P10832), ISH1_STOHE (P31713), ISH2_STOHE (P81129),ISIK_HELPO (P00994), ISP2_GALME (P81906), IVB1_BUNFA (P25660),IVB1_BUNMU (P00987), IVB1_VIPAA (P00991), IVB2_BUNMU (P00989),IVB2_DABRU (P00990), IVB2_HEMHA (P00985), IVB2_NAJNI (P00986),IVB3_VIPAA (P00992), IVBB_DENPO (P00983), IVBC_NAJNA (P19859),IVBC_OPHHA (P82966), IVBE_DENPO (P00984), IVBI_DENAN (P00980),IVBI_DENPO (P00979), IVBK_DENAN (P00982), IVBK_DENPO (P00981),IVBT_ERIMA (P24541), IVBT_NAJNA (P20229), MCPI_MELCP (P82968),SBPI_SARBU (P26228), SPT3_HUMAN (P49223), TKD1_BOVIN (Q28201),TKD1_SHEEP (Q29428), TXCA_DENAN (P81658), UPTI_PIG (Q29100), AMBP_BOVIN(P00978), AMBP_HUMAN (P02760), AMBP_MERUN (Q62577), AMBP_MESAU (Q60559),AMBP_MOUSE (Q07456), AMBP_PIG (P04366), AMBP_RAT (Q64240), IATR_HORSE(P04365), IATR_SHEEP (P13371), SPT1_HUMAN (O43278), SPT1_MOUSE (Q9R097),SPT2_HUMAN (O43291), SPT2_MOUSE (Q9WU03), TFP2_HUMAN (P48307),TFP2_MOUSE (O35536), TFPI_HUMAN (P10646), TFPI_MACMU (Q28864),TFPI_MOUSE (O54819), TFPI_RABIT (P19761), TFPI_RAT (Q02445), YN81_CAEEL(Q03610)

A variety of methods can be used to identify a Kunitz domain from asequence database. For example, a known amino acid sequence of a Kunitzdomain, a consensus sequence, or a motif (e.g., the ProSite Motif) canbe searched against the GenBank sequence databases (National Center forBiotechnology Information, National Institutes of Health, Bethesda Md.),e.g., using BLAST; against Pfam database of HMMs (Hidden Markov Models)(e.g., using default parameters for Pfam searching; against the SMARTdatabase; or against the ProDom database. For example, the PfamAccession Number PF00014 of Pfam Release 9 provides numerous Kunitzdomains and an HMM for identify Kunitz domains. A description of thePfam database can be found in Sonhammer et al. (1997) Proteins28(3):405-420 and a detailed description of HMMs can be found, forexample, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskovet al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz el al. (1993) ProteinSci. 2:305-314. The SMART database (Simple Modular Architecture ResearchTool. EMBL, Heidelberg, Del.) of HMMs as described in Schultz et at(1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (2000)Nucl. Acids Res 28:231. The SMART database contains domains identifiedby profiling with the hidden Markov models of the HMMer2 search program(R. Durbin et al. (1998) Biological sequence analysis: probabilisticmodels of proteins and nucleic acids. Cambridge University Press). Thedatabase also is annotated and monitored. The ProDom protein domaindatabase consists of an automatic compilation of homologous domains(Corpet et al. (1999). Nucl. Acids Res. 27:263-267). Current versions ofProDom are built using recursive PSI-BLAST searches (Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computersand Chemistry 23:333-340) of the SWISS-PROT 38 and TREMBL proteindatabases. The database automatically generates a consensus sequence foreach domain. Prosite lists the Kunitz domain as a motif and identifiesproteins that include a Kunitz domain. See, e.g., Falquet et al. NucleicAcids Res. 30:235-238(2002).

Kunitz domains interact with target protease using, primarily, aminoacids in two loop regions (“binding loops”). The first loop region isbetween about residues corresponding to amino acids 13-20 of BPTI. Thesecond loop region is between about residues corresponding to aminoacids 31-39 of BPTI. An exemplary library of Kunitz domains varies oneor more amino acid positions in the first and/or second loop regions.Particularly useful positions to vary, when screening for Kunitz domainsthat interact with kallikrein or when selecting for improved affinityvariants, include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39with respect to the sequence of BPTI. At least some of these positionsare expected to be in close contact with the target protease. It is alsouseful to vary other positions, e.g., positions that are adjacent to theaforementioned positions in the three-dimensional structure.

The “framework region” of a Kunitz domain is defined as those residuesthat are a part of the Kunitz domain, but specifically excludingresidues in the first and second binding loops regions, i.e., aboutresidues corresponding to amino acids 13-20 of BPTI and 31-39 of BPTI.Conversely, residues that are not in the binding loop may tolerate awider range of amino acid substitution (e.g., conservative and/ornon-conservative substitutions).

In one embodiment, these Kunitz domains are variant forms of the loopedstructure including Kunitz domain 1 of human lipoprotein-associatedcoagulation inhibitor (LACI) protein. LACI contains three internal,well-defined, peptide loop structures that are paradigm Kunitz domains(Girard, T. et al., 1989. Nature, 338:518-520). Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287). These methods can also beapplied to other Kunitz domain frameworks to obtain other Kunitz domainsthat interact with kallikrein, e.g., plasma kallikrein. Usefulmodulators of kallikrein function typically bind and/or inhibitkallikrein, as determined using kallikrein binding and inhibitionassays.

An exemplary polypeptide that includes a Kunitz domain that inhibitsplasma kallikrein has or includes the amino acid sequence defined byamino acids 3-60 of SEQ ID NO:2. Another exemplary polypeptide thatincludes a Kunitz domain that inhibits plasma kallikrein has or includesthe amino acid sequence of SEQ ID NO:2.

An exemplary polypeptide includes the amino acid sequence:

(SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53Xaa54 Cys Xaa56 Xaa57 Xaa58.

“Xaa” refers to a position in a peptide chain that can be any of anumber of different amino acids. In a first example, Xaa can by anyamino acid except cysteine. In another example, one or more of thefollowing apply: Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gin; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gin or Thr, Xaa18 can be His, Leu, Gin or Ala; Xaa19 canbe Pro, Gin, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gin, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32can be Glu, Gin, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54can be any amino acid.

Additionally, each of the first four (Xaa1, Xaa2, Xaa3. Xaa4) and atlast three 9 Xaa56, Xaa57 or Xaa58) amino acids of SEQ ID NO: 1 canoptionally be present or absent and can be any amino acid, if present,e.g., any non-cysteine amino acid

In one embodiment, the polypeptide has a sequence with one or more ofthe following properties: Xaa11 can be Asp, Gly, Ser or Val; Xaa13 canbe Pro, Arg, His or Asn; Xaa15 can be Arg or Lys; Xaa16 can be Ala orGly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18 can be His, Leu or Gin;Xaa19 can be Pro. Gin or Leu; Xaa21 can be Trp or Phe; Xaa31 is Glu;Xaa32 can be Glu or Gin; Xaa34 can be lie, Thr or Ser, Xaa35 is Tyr, andXaa39 can be Glu, Gly or Ala.

An exemplary polypeptide can include the following amino acids: Xaa10 isAsp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can beAla or Gly; Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp;Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile or Ser, Xaa35 is Tyr; andXaa39 is Gly.

It is also possible to use portions of the polypeptides describedherein. For example, polypeptides could include binding domains forspecific kallikrein epitopes. For example, the binding loops of Kunitzdomains can by cyclized and used in isolation or can be grafted ontoanother domain, e.g., a framework of another Kunitz domain. It is alsopossible to remove one, two, three, or four amino acids from theN-terminus of an amino acid sequence described herein, and/or one, two,three, four, or five amino acids from the C-terminus of an amino acidsequence described herein.

Additional examples of sequence include those that differ by at leastone amino acid, but fewer than seven, six, five, four, three, or twoamino acids differences relative to an amino acid sequence describedherein, e.g., an amino acid sequence provided above. In one embodiment,fewer than three, two, or one differences are in one of the bindingloops. For example, the first binding loop may have no differencesrelative to an amino acid sequence described herein, e.g., an amino acidsequence provided above. In another example, neither the first nor thesecond binding loop differs from an amino acid sequence describedherein, e.g., an amino acid sequence provided above.

Still others polypeptides that inhibit plasma kallikrein include anabout 58-amino acid sequence of amino acids 3-60 of SEQ ID NO:2 or thePEP-1 polypeptide having the 60-amino acid sequence of SEQ ID NO:2. Theterms “PEP-1” and “DX-88” as used herein both refer to the 60-amino acidsequence of SEQ ID NO:2. In one embodiment, the polypeptide is otherthan aprotinin, e.g., differs from aprotinin, by at least one, two,three, five, ten, or fifteen amino acids.

Polypeptides described herein can be made synthetically using anystandard polypeptide synthesis protocol and equipment. For example, thestepwise synthesis of a polypeptide can be carried out by the removal ofan amino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the I-amino protecting group, and theFMOC method, which utilizes 9-fluorenylmethloxycarbonyl to protect thealpha-amino of the amino acid residues. Both methods are well known tothose of skill in the art (Stewart, J, and Young, J., Solid-PhasePeptide Synthesis (W. H. Freeman Co., San Francisco 1989); Merrifield,J., 1963. Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A.,The Practice of Peptide Synthesis (Springer-Verlag, New York 1984)). Ifdesired, additional amino- and/or carboxy-terminal amino acids can bedesigned into the amino acid sequence and added during polypeptidesynthesis.

Polypeptides can also be produced using recombinant technology.Recombinant methods can employ any of a number of cells andcorresponding expression vectors, including but not limited to bacterialexpression vectors, yeast expression vectors, baculovirus expressionvectors, mammalian viral expression vectors, and the like. A polypeptidedescribed herein can be produced by a transgenic animal, e.g., in themammary gland of a transgenic animal. In some cases, it could benecessary or advantageous to fuse the coding sequence for a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) to another coding sequence in an expression vector to form afusion polypeptide that is readily expressed in a host cell. Part or allof the additional sequence can be removed, e.g., by protease digestion.

An exemplary recombinant expression system for producing a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) is a yeast expression vector, which permits a nucleic acidsequence encoding the amino acid sequence for the inhibitor polypeptideto be linked in the same reading frame with a nucleotide sequenceencoding the MATα prepro leader peptide sequence of Saccharomycescerevisiae, which in turn is under the control of an operable yeastpromoter. The resulting recombinant yeast expression plasmid can betransformed by standard methods into the cells of an appropriate,compatible yeast host, which cells are able to express the recombinantprotein from the recombinant yeast expression vector. Preferably, a hostyeast cell transformed with such a recombinant expression vector is alsoable to process the fusion protein to provide an active inhibitorpolypeptide. An other exemplary yeast host for producing recombinantpolypeptides is Pichia pastoris.

As noted above, polypeptides that inhibit kallikrein can include aKunitz domain polypeptide described herein. Some polypeptides caninclude an additional flanking sequence, preferably of one to six aminoacids in length, at the amino and/or carboxy-terminal end, provided suchadditional amino acids do not significantly diminish kallikrein bindingaffinity or kallikrein inhibition activity so as to preclude use in themethods and compositions described herein. Such additional amino acidscan be deliberately added to express a polypeptide in a particularrecombinant host cell or can be added to provide an additional function,e.g., to provide a linker to another molecule or to provide an affinitymoiety that facilitates purification of the polypeptide. Preferably, theadditional amino acid(s) do not include cysteine, which could interferewith the disulfide bonds of the Kunitz domain.

An exemplary Kunitz domain polypeptide includes the amino acid sequenceof residues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with theMATalpha-prepro leader peptide sequence of S. cerevisiae. When secretedfrom the yeast host cell, most of the leader peptide is processed fromthe fusion protein to yield a functional polypeptide (referred to hereinas “PEP-1”) having the amino acid sequence of SEQ ID NO:2.

A typical Kunitz domain, e.g., that includes, SEQ ID NO: 1, contains anumber of invariant positions, e.g., positions corresponding to position5, 14, 30, 33, 38, 45, 51 and 55 in the BPTI numbering scheme arecysteine. The spacing between these positions may vary to the extentallowable within the Kunitz domain fold, e.g., such that three disulfidebonds are formed. Other positions such as, for example, positions 6, 7,8, 9, 20, 24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53and 54, or positions corresponding to those positions, can be any aminoacid (including non-genetically encoded occurring amino acids). In aparticularly preferred embodiment, one or more amino acids correspond tothat of a native sequence. In another embodiment, at least one variableposition is different from that of the native sequence. In yet anotherpreferred embodiment, the amino acids can each be individually orcollectively substituted by a conservative or non-conservative aminoacid substitution.

Conservative amino acid substitutions replace an amino acid with anotheramino acid of similar chemical nature and may have no affect on proteinfunction. Non-conservative amino acid substitutions replace an aminoacid with another amino acid of dissimilar chemical structure. Examplesof conserved amino acid substitutions include, for example, Asn→Gln,Arg→Lys and Ser→Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2.

Other positions, for example, positions 10, 11, 13, 15, 16, 17, 18, 19,21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or positionscorresponding to those positions can be any of a selected set of aminoacids. For example, SEQ ID NO: 1 defines a set of possible sequences.Each member of this set contains, for example, a cysteine at positions5, 14, 30, 51 and 55, and any one of a specific set of amino acids atpositions 10, 11, 13, 15, 16, 17, 18, 19, 21, 22, 23, 31, 32, 34, 35,39, 40, 43 and 45, or positions corresponding to those positions. In apreferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 and/or 19 of these amino acids can be independently orcollectively, in any combination, selected to correspond to thecorresponding position of SEQ ID NO:2. The polypeptide preferably has atleast 80%, 85%, 90%, 95, 97, 98, or 99% identity to SEQ ID NO:2.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444-453, algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about what parameters should be applied todetermine if a molecule is within a homology limitation) are a Blossum62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4,and a frameshift gap penalty of 5.

Binding Protein Inhibitors.

In other embodiments, the inhibitors of kallikrein are binding proteins,such as antibodies. Exemplary binding proteins such as antibodies aredescribed, e.g., in PCT Publication WO2012/094587 and US PatentApplication Publication US 20100183625, both of which are incorporatedherein by reference in their entirety.

In one aspect, the disclosure features a protein (e.g., an isolatedprotein) that binds to plasma kallikrein (e.g., human plasma kallikrein)and includes at least one immunoglobulin variable region. For example,the protein includes a heavy chain (HC) immunoglobulin variable domainsequence and/or a light chain (LC) immunoglobulin variable domainsequence. The protein can bind to and inhibit plasma kallikrein, e.g.,human plasma kallikrein.

The protein can include one or more of the following characteristics:(a) a human CDR or human framework region; (b) the HC immunoglobulinvariable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRsthat are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identical to a CDR of a HC variable domain described herein; (c)the LC immunoglobulin variable domain sequence comprises one or more(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variabledomain described herein; (d) the LC immunoglobulin variable domainsequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or 100% identical to a LC variable domain described herein (e.g.,overall or in framework regions or CDRs); (e) the HC immunoglobulinvariable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identical to a HC variable domain describedherein (e.g., overall or in framework regions or CDRs); (f) the proteinbinds an epitope bound by a protein described herein, or competes forbinding with a protein described herein; (g) a primate CDR or primateframework region; (h) the HC immunoglobulin variable domain sequencecomprises a CDR1 that differs by at least one amino acid but by no morethan 2 or 3 amino acids from the CDR1 of a HC variable domain describedherein; (i) the HC immunoglobulin variable domain sequence comprises aCDR2 that differs by at least one amino acid but by no more than 2, 3,4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domaindescribed herein; (j) the HC immunoglobulin variable domain sequencecomprises a CDR3 that differs by at least one amino acid but by no morethan 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variable domaindescribed herein; (k) the LC immunoglobulin variable domain sequencecomprises a CDR1 that differs by at least one amino acid but by no morethan 2, 3, 4, or 5 amino acids from the CDR1 of a LC variable domaindescribed herein; (1) the LC immunoglobulin variable domain sequencecomprises a CDR2 that differs by at least one amino acid but by no morethan 2, 3, or 4 amino acids from the CDR2 of a LC variable domaindescribed herein; (m) the LC immunoglobulin variable domain sequencecomprises a CDR3 that differs by at least one amino acid but by no morethan 2, 3, 4, or 5 amino acids from the CDR3 of a LC variable domaindescribed herein; (n) the LC immunoglobulin variable domain sequencediffers by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids from a LC variable domain described herein(e.g., overall or in framework regions or CDRs); and (o) the HCimmunoglobulin variable domain sequence differs by at least one aminoacid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids froma HC variable domain described herein (e.g., overall or in frameworkregions or CDRs).

The plasma kallikrein binding protein may be an isolated protein (e.g.,at least 70, 80, 90, 95, or 99% free of other proteins). The plasmakallikrein binding protein may inhibit plasma kallikrein, e.g., humanplasma kallikrein. In some embodiments, the plasma kallikrein bindingprotein does not bind prekallikrein (e.g., human prekallikrein), butbinds to the active form of plasma kallikrein (e.g., human plasmakallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasmakallikrein. In some aspects, the protein binds the same epitope orcompetes for binding with a protein described herein.

In some embodiments, the protein competes with or binds the same epitopeas M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred toherein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03,X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,X115-F02, X124-G01 (also referred to herein as DX-2930), X115-G04,M29-D09, M145-D11, M06-D09 and M35-G04. See, e.g., PCT PublicationWO2012/094587 and US Patent Application Publication US 20100183625 Insome embodiments, the protein binds to one or more amino acids that formthe catalytic triad of plasma kallikrein: His434. Asp483, and/or Ser578(numbering based on the human sequence).

In some embodiments, the protein binds to one or more amino acids ofSer479. Tyr563, and/or Asp585 (numbering based on the human sequence).

The protein can bind to plasma kallikrein, e.g., human plasmakallikrein, with a binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰ and 10¹¹ M⁻¹. In one embodiment, the protein binds to human plasmakallikrein with a K_(off) slower than 1×10⁻³, 5×10⁻⁴ s⁻¹, or 1×10⁻⁴ s⁻¹.In one embodiment, the protein binds to human plasma kallikrein with aK_(on) faster than 1×10², 1×10³, or 5×10³ M⁻¹s⁻¹. In one embodiment, theprotein binds to plasma kallikrein, but does not binds to tissuekallikrein and/or plasma prekallikrein (e.g., the protein binds totissue kallikrein and/or plasma prekallikrein less effectively (e.g.,5-, 10-, 50-, 100-, or 1000-fold less or not at all, e.g., as comparedto a negative control) than it binds to plasma kallikrein.

In one embodiment, the protein inhibits human plasma kallikreinactivity, e.g., with a Ki of less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, and10⁻¹⁰ M. The protein can have, for example, an IC50 of less than 100 nM,10 nM or 1 nM. For example, the protein may modulate plasma kallikreinactivity, as well as the production of Factor XIIa (e.g., from FactorXII) and/or bradykinin (e.g., from high-molecular-weight kininogen(HMWK)). The protein may inhibit plasma kallikrein activity, and/or theproduction of Factor XIIa (e.g., from Factor XII) and/or bradykinin(e.g., from high-molecular-weight kininogen (HMWK)). The affinity of theprotein for human plasma kallikrein can be characterized by a K_(D) ofless than 100 nm, less than 10 nM, or less than 1 nM. In one embodiment,the protein inhibits plasma kallikrein, but does not inhibit tissuekallikrein (e.g., the protein inhibits tissue kallikrein lesseffectively (e.g., 5-, 10-, 50-, 100-, or 1000-fold less or not at all,e.g., as compared to a negative control) than it inhibits plasmakallikrein.

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, or 10 nM.

Plasma kallikrein binding proteins may be antibodies. Plasma kallikreinbinding antibodies may have their HC and LC variable domain sequencesincluded in a single polypeptide (e.g., scFv), or on differentpolypeptides (e.g., IgG or Fab).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and/or heavy chains of antibodies selectedfrom the group consisting of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03,X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred toherein as DX-2930), X115-G104, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, MI 60-G12, M142-H08, X63-G06, X101-A01 (alsoreferred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930),X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRsselected from the corresponding CDRs of the group of light chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (alsoreferred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930),X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs and oneor more (e.g., 1, 2, or 3) light chain CDRs selected from thecorresponding CDRs of the group of light chains consisting of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein asDX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X124-G01 (also referred to herein as DX-2930), X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain. In another, the HC and LCvariable domain sequences are components of different polypeptidechains. For example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, orIgG4. The protein can be a soluble Fab. In other implementations theprotein includes a Fab2′, scFv, minibody, scFv::Fc fusion, Fab::HSAfusion. HSA::Fab fusion. Fab::HSA::Fab fusion, or other molecule thatcomprises the antigen combining site of one of the binding proteinsherein. The VH and VL regions of these Fabs can be provided as IgG, Fab.Fab2, Fab2′, scFv, PEGylated Fab. PEGylated scFv, PEGylated Fab2.VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, orother appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to human framework regions. In oneembodiment, the protein includes a human Fc domain, or an Fe domain thatis at least 95, 96, 97, 98, or 99% identical to a human Fc domain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to primate framework regions. In oneembodiment, the protein includes a primate Fc domain, or an Fc domainthat is at least 95, 96, 97, 98, or 99% identical to a primate Fcdomain. “Primate” includes humans (Homo sapiens), chimpanzees (Pantroglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla),gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), andtarsiers.

In some embodiments, the affinity of the primate antibody for humanplasma kallikrein is characterized by a K_(D) of less than 1000, 500,100 or 10 nM, e.g., less than 10 nM or less than 1 nM.

In one embodiment, the protein includes human framework regions, orframework regions that are at least 95, 96, 97, 98, or 99% identical tohuman framework regions. In certain embodiments, the protein includes nosequences from mice or rabbits (e.g., is not a murine or rabbitantibody).

In some aspects, the disclosure provides the use of proteins (e.g.,binding proteins, e.g., antibodies) (e.g., the proteins describedherein) that bind to plasma kallikrein (e.g., human plasma kallikrein)and include at least one immunoglobin variable region in methods fortreating a disease or disorder described herein. For example, the plasmakallikrein binding protein includes a heavy chain (HC) immunoglobulinvariable domain sequence and a light chain (LC) immunoglobulin variabledomain sequence. A number of exemplary plasma kallikrein bindingproteins are described herein.

Antibodies may be discovered by screening a library using a kallikreintarget, as well as by other methods. For example, kallikrein protein ora region thereof can be used as an antigen in a non-human animal, e.g.,a rodent. Humanized antibodies can be generated by replacing sequencesof the Fv variable region that are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General methods for generating humanized antibodies are provided byMorrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089,5,693,761 and 5,693,762. Those methods include isolating, manipulating,and expressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Numerous sources of such nucleic acid are available. For example,nucleic acids may be obtained from a hybridoma producing an antibodyagainst a predetermined target, as described above. The recombinant DNAencoding the humanized antibody, or fragment thereof, can then be clonedinto an appropriate expression vector.

Immunoglobin kallikrein binding proteins (e.g., IgG or Fab kallikreinbinding proteins) may be modified to reduce immunogenicity. Reducedimmunogenicity is desirable in kallikrein binding proteins intended foruse as therapeutics, as it reduces the chance that the subject willdevelop an immune response against the therapeutic molecule. Techniquesuseful for reducing immunogenicity of kallikrein binding proteinsinclude deletion/modification of potential human T cell epitopes and‘germlining’ of sequences outside of the CDRs (e.g., framework and Fc).

A kallikrein-binding antibody may be modified by specific deletion ofhuman T cell epitopes or “deimmunization” by the methods disclosed in WO98/52976 and WO 00/34317. Briefly, the heavy and light chain variableregions of an antibody are analyzed for peptides that bind to MHC ClassII; these peptides represent potential T-cell epitopes (as defined in WO98/52976 and WO 00/34317). For detection of potential T-cell epitopes, acomputer modeling approach termed “peptide threading” can be applied,and in addition a database of human MHC class 11 binding peptides can besearched for motifs present in the VH and VL sequences, as described inWO 98/52976 and WO 00/34317. These motifs bind to any of the 18 majorMHC class II DR allotypes, and thus constitute potential T cellepitopes. Potential T-cell epitopes detected can be eliminated bysubstituting small numbers of amino acid residues in the variableregions, or preferably, by single amino acid substitutions. As far aspossible conservative substitutions are made, often but not exclusively,an amino acid common at this position in human germline antibodysequences may be used. Human germline sequences are disclosed inTomlinson, I. A. et al., 1992, J. Mol. Biol. 227:776-798; Cook, G. P. etal., 1995, Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al.,1992, J. Mol. Bio. 227:799-817. The V BASE directory provides acomprehensive directory of human immunoglobulin variable regionsequences (compiled by Tomlinson, I. A. et al. MRC Centre for ProteinEngineering, Cambridge, UK). After the deimmunizing changes areidentified, nucleic acids encoding V_(H) and V_(L) can be constructed bymutagenesis or other synthetic methods (e.g., de novo synthesis,cassette replacement, and so forth). Mutagenized variable sequence can,optionally, be fused to a human constant region, e.g., human IgG1 or Kconstant regions.

In some cases a potential T cell epitope will include residues which areknown or predicted to be important for antibody function. For example,potential T cell epitopes are usually biased towards the CDRs. Inaddition, potential T cell epitopes can occur in framework residuesimportant for antibody structure and binding. Changes to eliminate thesepotential epitopes will in some cases require more scrutiny, e.g., bymaking and testing chains with and without the change. Where possible,potential T cell epitopes that overlap the CDRs were eliminated bysubstitutions outside the CDRs. In some cases, an alteration within aCDR is the only option, and thus variants with and without thissubstitution should be tested. In other cases, the substitution requiredto remove a potential T cell epitope is at a residue position within theframework that might be critical for antibody binding. In these cases,variants with and without this substitution should be tested. Thus, insome cases several variant deimmunized heavy and light chain variableregions were designed and various heavy/light chain combinations testedin order to identify the optimal deimmunized antibody. The choice of thefinal deimmunized antibody can then be made by considering the bindingaffinity of the different variants in conjunction with the extent ofdeimmunization, i.e., the number of potential T cell epitopes remainingin the variable region. Deimmunization can be, used to modify anyantibody, e.g., an antibody that includes a non-human sequence, e.g., asynthetic antibody, a murine antibody other non-human monoclonalantibody, or an antibody isolated from a display library.

Kallikrein binding antibodies are “germlined” by reverting one or morenon-germline amino acids in framework regions to corresponding germlineamino acids of the antibody, so long as binding properties aresubstantially retained. Similar methods can also be used in the constantregion, e.g., in constant immunoglobulin domains.

Antibodies that bind to kallikrein, e.g., an antibody described herein,may be modified in order to make the variable regions of the antibodymore similar to one or more germline sequences. For example, an antibodycan include one, two, three, or more amino acid substitutions, e.g., ina framework, CDR, or constant region, to make it more similar to areference germline sequence. One exemplary germlining method can includeidentifying one or more germline sequences that are similar (e.g., mostsimilar in a particular database) to the sequence of the isolatedantibody. Mutations (at the amino acid level) are then made in theisolated antibody, either incrementally or in combination with othermutations. For example, a nucleic acid library that includes sequencesencoding some or all possible germline mutations is made. The mutatedantibodies are then evaluated, e.g., to identify an antibody that hasone or more additional germline residues relative to the isolatedantibody and that is still useful (e.g., has a functional activity). Inone embodiment, as many germline residues are introduced into anisolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a framework and/or constant region. Forexample, a germline framework and/or constant region residue can be froma germline sequence that is similar (e.g., most similar) to thenon-variable region being modified. After mutagenesis, activity (e.g.,binding or other functional activity) of the antibody can be evaluatedto determine if the germline residue or residues are tolerated (i.e., donot abrogate activity). Similar mutagenesis can be performed in theframework regions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may includingusing two germline sequences that separately contribute to theamino-terminal portion and the carboxy-terminal portion. In otherimplementations more than one or two germline sequences are used, e.g.,to form a consensus sequence.

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% ofthe CDR amino acid positions that are not identical to residues in thereference CDR sequences, residues that are identical to residues atcorresponding positions in a human germline sequence (i.e., an aminoacid sequence encoded by a human germline nucleic acid).

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 50, 60, 70, 80, 90 or 100% of the FRregions identical to FR sequence from a human germline sequence, e.g., agermline sequence related to the reference variable domain sequence.

Accordingly, it is possible to isolate an antibody which has similaractivity to a given antibody of interest, but is more similar to one ormore germline sequences, particularly one or more human germlinesequences. For example, an antibody can be at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 99.5% identical to a germline sequence in aregion outside the CDRs (e.g., framework regions). Further, an antibodycan include at least 1, 2, 3, 4, or 5 germline residues in a CDR region,the germline residue being from a germline sequence of similar (e.g.,most similar) to the variable region being modified. Germline sequencesof primary interest are human germline sequences. The activity of theantibody (e.g., the binding activity as measured by K_(A)) can be withina factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.

Germline sequences of human immunoglobin genes have been determined andare available from a number of sources, including the internationalImMunoGeneTics information System® (IMGT), available via the world wideweb at imgt.cines.fr, and the V BASE directory (compiled by Tomlinson,I. A. et al. MRC Centre for Protein Engineering, Cambridge, UK,available via the world wide web at vbase.mrc-cpe.cam.ac.uk).

Exemplary germline reference sequences for V_(kappa) include: O12/O2,O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23, L9, L24, L11,L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11, L2/L116, L6, L20,L25, B3, B2, A26/A10, and A14. See, e.g., Tomlinson et al., 1995. EMBOJ. 14(18):4628-3.

A germline reference sequence for the HC variable domain can be based ona sequence that has particular canonical structures, e.g., 1-3structures in the H1 and H2 hypervariable loops. The canonicalstructures of hypervariable loops of an immunoglobulin variable domaincan be inferred from its sequence, as described in Chothia et al., 1992,J. Mol. Biol. 227:799-817; Tomlinson et al., 1992, J. Mol. Biol.227:776-798); and Tomlinson et al., 1995, EMBO J. 14(18):4628-38.Exemplary sequences with a 1-3 structure include: DP-1, DP-8, DP-12,DP-2, DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2,hv3005, hv3005f3, DP-46, DP-47, DP-58, DP-49, DP-50, DP-51, DP-53, andDP-54.

Useful polypeptides can also be encoded by a nucleic acid thathybridizes to a nucleic acid that encodes a polypeptide describedherein. The nucleic acids can hybridize under medium, high, or very highstringency conditions. As used herein, the term “hybridizes under lowstringency, medium stringency, high stringency, or very high stringencyconditions” describes conditions for hybridization and washing. Guidancefor performing hybridization reactions can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, whichis incorporated by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C.

Protein Production.

Standard recombinant nucleic acid methods can be used to express aprotein that binds to plasma kallikrein. Generally, a nucleic acidsequence encoding the protein is cloned into a nucleic acid expressionvector. Of course, if the protein includes multiple polypeptide chains,each chain can be cloned into an expression vector, e.g., the same ordifferent vectors, that are expressed in the same or different cells.

Antibody Production.

Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g.,E. coli cells. For example, if the Fab is encoded by sequences in aphage display vector that includes a suppressible stop codon between thedisplay entity and a bacteriophage protein (or fragment thereof), thevector nucleic acid can be transferred into a bacterial cell that cannotsuppress a stop codon. In this case, the Fab is not fused to the gene IIprotein and is secreted into the periplasm and/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment,the antibodies (e.g., scFv's) are expressed in a yeast cell such asPichia (see, e.g., Powers et al., 2001, J. Immunol. Methods.251:123-35), Hanseula, or Saccharomyces.

In one preferred embodiment, antibodies are produced in mammalian cells.Preferred mammalian host cells for expressing the clone antibodies orantigen-binding fragments thereof include Chinese Hamster Ovary (CHOcells) (including dhfr-CHO cells, described in Urlaub and Chasin, 1980,Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601 621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2cells, COS cells, HEK293T cells (J. Immunol. Methods (2004)289(1-2):65-80), and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the diversifiedimmunoglobulin domain, the recombinant expression vectors may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

In an exemplary system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked toenhancer/promoter regulatory elements (e.g., derived from SV40, CMV,adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carries a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G coupled matrix.

For antibodies that include an Fc domain, the antibody production systemmay produce antibodies in which the Fc region is glycosylated. Forexample, the Fc domain of IgG molecules is glycosylated at asparagine297 in the CH2 domain. This asparagine is the site for modification withbiantennary-type oligosaccharides. It has been demonstrated that thisglycosylation is required for effector functions mediated by Fcgreceptors and complement C1q (Burton and Woof, 1992, Adv. Immunol.51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In oneembodiment, the Fc domain is produced in a mammalian expression systemthat appropriately glycosylates the residue corresponding to asparagine297. The Fc domain can also include other eukaryotic post-translationalmodifications.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method of expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acids encoding theantibody of interest and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody can be purifiedfrom the milk, or for some applications, used directly.

(C) Modifications

It is possible to modify polypeptides that inhibit kallikrein in avariety of ways. For example, the polypeptides can be attached to one ormore polyethylene glycol moieties to stabilize the compound or prolongretention times, e.g., by at least 2, 4, 5, 8, 10, 15, 20, 50, 100, 500or 1000 fold.

In one embodiment, a kallikrein binding protein is physically associatedwith a moiety that improves its stabilization and/or retention incirculation, e.g., in blood, serum, lymph, or other tissues, e.g., by atleast 1.5, 2, 5, 10, or 50 fold. For example, a kallikrein bindingprotein can be associated with a polymer, e.g., a substantiallynon-antigenic polymer, such as a polyalkylene oxide or polyethyleneoxide. Suitable polymers will vary substantially by weight. Polymershaving molecular number average weights ranging from about 200 to about35,000 (or about 1,000 to about 15.000, and 2,000 to about 12.500) canbe used. For example, a kallikrein binding protein can be conjugated toa water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.polyvinylalcohol and polyvinylpyrrolidone. A plurality of polymermoieties can be attached to one polypeptide, e.g., at least two, three,or four such moieties, e.g., having an average molecular weight of about2.000 to 7.000 Daltons. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained.

For example, the polypeptide can be conjugated to a water solublepolymer, e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcoholand polyvinylpyrrolidone. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained. Additional useful polymers includepolyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and blockcopolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid). D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextranesulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparan.

It is possible for one or more framework and/or CDR amino acid residuesof a binding protein to include one or more mutations (e.g.,substitutions (e.g., conservative substitutions or substitutions ofnon-essential amino acids), insertions, or deletions) relative to abinding protein described herein. A plasma kallikrein binding proteinmay have mutations (e.g., substitutions (e.g., conservativesubstitutions or substitutions of non-essential amino acids),insertions, or deletions) (e.g., at least one, two, three, or four,and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations)relative to a binding protein described herein, e.g., mutations which donot have a substantial effect on protein function. The mutations can bepresent in framework regions, CDRs, and/or constant regions. In someembodiments, the mutations are present in a framework region. In someembodiments, the mutations are present in a CDR. In some embodiments,the mutations are present in a constant region. Whether or not aparticular substitution will be tolerated, i.e., will not adverselyaffect biological properties, such as binding activity can be predicted,e.g., by evaluating whether the mutation is conservative or by themethod of Bowie, et al. (1990) Science 247:1306-1310.

A kallikrein inding protein can also be associated with a carrierprotein, e.g., a serum albumin, such as a human serum albumin. Forexample, a translational fusion can be used to associate the carrierprotein with the kallikrein binding protein.

(D) Treating Auto-Immune Diseases Associated with Kallikrein System

One or more of the pKal inhibitors as described herein can be used totreating diseases associated with the pKal system, including, but notlimited to diabetic macular edema, retinal proliferation, brain trauma,acute spinal cord injury, localized amyloidosis, autoimmune diseasessuch as psoriasis, multiple aclerosis, inflammatory bowel disease,rheumatoid arthritis, vasculitis, systemic lupus erythematosisnephritis, systemic mastocytosis, severe burns, and neuropathic pain(diabetic and post-herpetic neuralgia).

A subject who is at risk (e.g., a human patient) for developing anautoimmune or the other pKal-associated diseases mentioned herein canbe, e.g., a subject who has a disease associated with the development ofthe disease, a subject who has been exposed to an environmental factorassociated with the development of the disease, a subject who has afamily history of the disease, or a subject who carries a geneassociated with the development of the disease.

The subjects can be humans in need of treatment for an autoimmune or oneof the other pKal-associated disease (e.g., humans having the disease orat risk of developing the disease) or nonhuman subjects (e.g., an animalmodel of an autoimmune or one of the other pKal-associated disease).

In some embodiments, the subject is a subject (e.g., a human patient)who is at risk for developing an autoimmune disease such as rheumatoidarthritis (RA), Crohn's disease (CD) or Ulcerative colitis (UC).Autoimmune diseases are diseases caused by an abnormal immune responseto a subject's own body. The abnormal immune response may be against acertain organ or tissue, depending on the type of autoimmune disease.

RA is a chronic inflammatory disease generally affecting the joints,such as the synovial joints of the hands and/or feet. The inflammatoryresponse in RA often causes destruction of cartilage and fusion of thejoints, resulting in loss of function and mobility. Symptoms of RAinclude swollen and/or warm joints, stiffness, rheumatoid nodules,fatigue, fever, and weight loss. Exemplary treatments for RA includephysical therapy, orthoses, analgesics, anti-inflammatory drugs,steroids, and disease-modifying antirheumatic drugs (DMARDs).

CD is an inflammatory bowel disease that can affect any part of the GItract. Symptoms include abdominal pain, diarrhea, fever, fatigue andweight loss. Exemplary treatments for CD include corticosteroids,5-aminosalicylic acid drugs, azathioprine, methotrexate, infliximab,adalimumab, certolizumab, natalizumab, dietary adjustments, and surgery.

UC is an inflammatory bowel disease that generally affects the largeintestine. Symptoms include bloody and/or mucus-containing diarrhea,weight loss, anemia, abdominal pain, and blood in the rectum. Exemplarytreatments for UC include 5-aminosalicylic acid drugs, corticosteroids,azathioprine, budesonide, infliximab, adalimumab, and surgery.

(E) Combination Therapy

The plasma kallikrein inhibitor may be administered along with anothertherapeutic as part of a combination therapy for a disease or disorderdescribed herein.

Combination therapy with a kallikrein inhibitor and another therapeuticagent may be provided in multiple different configurations. Insituations where the kallikrein inhibitor is to be administered byintraarticular injection, the kallikrein inhibitor and the therapeuticagent may be co-administered as a single composition, or they may beadministered by separate injections. In some situations, the kallikreininhibitor and the therapeutic agent are administered in close temporalproximity (e.g., a short time interval between the injections, such asduring the same treatment session), or more widely spaced, depending onthe desired schedule of administration for the two components of thecombination therapy. When the kallikrein inhibitor is to be administeredby systemic (parenteral) administration, the kallikrein inhibitor andthe therapeutic agent may be administered in close temporal proximity ormore widely spaced, depending on the intended dosing schedule for thetwo components of the combination therapy.

In other embodiments, the kallikrein inhibitor may be administered incombination with other compounds useful for treating or preventinginflammation, which is involved in autoimmune diseases. Exemplaryanti-inflammatory agents include, for example, steroids (e.g., Cortisol,cortisone, fludrocortisone, prednisone, 6[alpha]-methylprednisone,triamcinolone, betamethasone or dexamethasone), nonsteroidalanti-inflammatory drugs (NSAIDS (e.g., aspirin, acetaminophen, tolmetin,ibuprofen, mefenamic acid, piroxicam, nabumetone, rofecoxib, celecoxib,etodolac or nimesulide). In another embodiment, the other therapeuticagent is an antibiotic (e.g., vancomycin, penicillin, amoxicillin,ampicillin, cefotaxime, ceftriaxone, cefixime, rifampinmetronidazole,doxycycline or streptomycin). In another embodiment, the othertherapeutic agent is a PDE4 inhibitor (e.g., roflumilast or rolipram).In another embodiment, the other therapeutic agent is an antihistamine(e.g., cyclizine, hydroxyzine, promethazine or diphenhydramine).

Further examples of anti-inflammatory agents include, for example,aceclofenac, acemetacin, e-acetamidocaproic acid, acetaminophen,acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine,alclofenac, alclometasone, alfentanil, algestone, allylprodine,alminoprofen, aloxiprin, alphaprodine, aluminum bis(acetylsalicylate),amcinonide, amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyricacid, 2-amino-4-picoline, aminopropylon, aminopyrine, amixetrine,ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine,antipyrine, antrafenine, apazone, beclomethasone, bendazac, benorylate,benoxaprofen, benzpiperylon, benzydamine, benzylmorphine, bermoprofen,betamethasone, betamethasone-17-valerate, bezitramide,[alpha]-bisabolol, bromfenac, p-bromoacetanilide, 5-bromosalicylic acidacetate, bromosaligenin, bucetin, bucloxic acid, bucolome, budesonide,bufexamac, bumadizon, buprenorphine, butacetin, butibufen, butorphanol,carbamazepine, carbiphene, caiprofen, carsalam, chlorobutanol,chloroprednisone, chlorthenoxazin, choline salicylate, cinchophen,cinmetacin, ciramadol, clidanac, clobetasol, clocortolone, clometacin,clonitazene, clonixin, clopirac, cloprednol, clove, codeine, codeinemethyl bromide, codeine phosphate, codeine sulfate, cortisone,cortivazol, cropropamide, crotethamide and cyclazocine.

Further examples of anti-inflammatory agents include deflazacort,dehydrotestosterone, desomorphine, desonide, desoximetasone,dexamethasone, dexamethasone-21-isonicotinate, dexoxadrol,dextromoramide, dextropropoxyphene, deoxycorticosterone, dezocine,diampromide, diamorphone, diclofenac, difenamizole, difenpiramide,ditlorasone, ditlucortolone, diflunisal, ditluprednate, dihydrocodeine,dihydrocodeinone enol acetate, dihydromorphine, dihydroxyaluminumacetylsalicylate, dimenoxadol, dimepheptanol, dimethylthiambutene,dioxaphetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazol,droxicam, emorfazone, enfenamic acid, enoxolone, epirizole, eptazocine,etersalate, ethenzamide, ethoheptazine, ethoxazene,ethylmethylthiambutene, ethylmorphine, etodolac, etofenamate,etonitazene, eugenol, felbinac, fenbufen, fenclozic acid, fendosal,fenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctafenine,fluazacort, flucloronide, flufenamic acid, flumethasone, flunisolide,flunixin, flunoxaprofen, fluocinolone acetonide, fluocinonide,fluocinolone acetonide, fluocortin butyl, fluocoitolone, fluoresone,fluorometholone, fluperolone, flupirtine, fluprednidene,fluprednisolone, fluproquazone, flurandrenolide, flurbiprofen,fluticasone, formocortal and fosfosal.

Further examples of anti-inflammatory agents include gentisic acid,glafenine, glucametacin, glycol salicylate, guaiazulene, halcinonide,halobetasol, halometasone, haloprednone, heroin, hydrocodone, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisonesuccinate, hydrocortisone hemisuccinate, hydrocortisone 21-lysinate,hydrocortisone cypionate, hydromorphone, hydroxypethidine, ibufenac,ibuprofen, ibuproxam, imidazole salicylate, indomethacin, indoprofen,isofezolac, isoflupredone, isoflupredone acetate, isoladol,isomethadone, isonixin, isoxepac, isoxicam, ketobemidone, ketoprofen,ketorolac, p-lactophenetide, lefetamine, levallorphan, levorphanol,levophenacyl-morphan, lofentanil, lonazolac, lornoxicam, loxoprofen,lysine acetylsalicylate, mazipredone, meclofenamic acid, medrysone,mefenamic acid, meloxicam, meperidine, meprednisone, meptazinol,mesalamine, metazocine, methadone, methotrimeprazine,methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, methylprednisolone suleptnate, metiazinic acid,metofoline, metopon, mofebutazone, mofezolac, mometasone, morazone,morphine, morphine hydrochloride, morphine sulfate, morpholinesalicylate and myrophine.

Further examples of anti-inflammatory agents include nabumetone,nalbuphine, nalorphine, 1-naphthyl salicylate, naproxen, narceine,nefopam, nicomorphine, nifenazone, niflumic acid, nimesulide,5′-nitro-2′-propoxyacetanilide, norlevorphanol, normethadone,normorphine, norpipanone, olsalazine, opium, oxaceprol, oxametacine,oxaprozin, oxycodone, oxymorphone, oxyphenbutazone, papaveretum,paramethasone, paranyline, parsalmide, pentazocine, perisoxal,phenacetin, phenadoxone, phenazocine, phenazopyridine hydrochloride,phenocoll, phenoperidine, phenopyrazone, phenomorphan, phenylacetylsalicylate, phenylbutazone, phenyl salicylate, phenyramidol,piketoprofen, piminodine, pipebuzone, piperylone, pirazolac,piritramide, piroxicam, pirprofen, pranoprofen, prednicarbate,prednisolone, prednisone, prednival, prednylidene, proglumetacin,proheptazine, promedol, propacetamol, properidine, propiram,propoxyphene, propyphenazone, proquazone, protizinic acid, proxazole,ramifenazone, remifentanil, rimazolium metilsulfate, salacetamide,salicin, salicylamide, salicylamide o-acetic acid, salicylic acid,salicylsulfuric acid, salsalate, salverine, simetride, sufentanil,sulfasalazine, sulindac, superoxide dismutase, suprofen, suxibuzone,talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine,thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine, tinoridine,tixocortol, tolfenamic acid, tolmetin, tramadol, triamcinolone,triamcinolone acetonide, tropesin, viminol, xenbucin, ximoprofen,zaltoprofen and zomepirac.

(F) Administration

The patient is generally a human, but may also be a non-human mammal.Human patients include adults, e.g., patients between ages 19-25, 26-40,41-55, 56-75, and 76 and older, and pediatric patients, e.g., patientsbetween ages 0-2, 3-6, 7-12, and 13-18.

The term “pharmaceutically acceptable” composition refers to a non-toxiccarrier or excipient that may be administered to a patient, togetherwith a kallikrein inhibitor described herein. The carrier or excipientis chosen to be compatible with the biological or pharmacologicalactivity of the composition. The kallikrein inhibitors (and, in the caseof combination therapy, other therapeutic agent) described herein can beadministered locally or systemically by any suitable means for deliveryof an inhibitory amount of the inhibitor and/or other therapeutic agentto a patient including but not limited to systemic administrations suchas, for example, intravenous and inhalation. Parenteral administrationis particularly preferred for the kallikrein inhibitor.

For parenteral administration, the kallikrein inhibitor can be injectedintravenously, intramuscularly, intraperitoneally, or subcutaneously.Subcutaneous injection and i.v. administration are preferred routes forparenteral administration. Also useful is local (intraarticular)injection.

Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer (e.g., sodium/potassium phosphatebuffered saline). Other pharmaceutically acceptable carriers include,but are not limited to, sterile water, saline solution, and bufferedsaline (including buffers like phosphate or acetate), alcohol, vegetableoils, polyethylene glycols, gelatin, lactose, amylose, magnesiumstearate, talc, silicic acid, paraffin, etc. Where necessary, thecomposition can also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection,preservatives, stabilizers, wetting agents, emulsifiers, salts,lubricants, etc. as long as they do not react deleteriously with theactive compounds. Similarly, the composition can comprise conventionalexcipients, e.g., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral or intranasalapplication which do not deleteriously react with the active compounds.Generally, the ingredients will be supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule, sachette, or vial indicating the quantity of active agent inactivity units. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade “water for injection” or saline. Where thecomposition is to be administered by injection, a container (e.g.,ampoule or vial) of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

Exemplary formulations for subcutaneous administration of an isolatedkallikrein inhibitor include buffered solutions containing a bufferingagent (e.g., histidine or phosphate buffer) and a cryoprotectant (e.g.,sucrose or sucrose and mannitol, optionally including a dextran such asdextran 40), and may be lyophilized for storage and distribution asdescribed in U.S. Pub. App. No. 2007-0213275 (U.S. Ser. No. 11/716,278,filed Mar. 9, 2007).

In one embodiment, the kallikrein inhibitor is administered to a patientas an intravenous infusion according to any approved procedure. Inanother embodiment, the kallikrein inhibitor is administered to apatient as a subcutaneous bolus. In another embodiment, the kallikreininhibitor is administered to a patient by intraarticular injection. I.V.and intraarticular administration are typically carried out by a healthcare professional in a clinical setting (e.g., hospital, urgent care, ordoctor's office), but subcutaneous injections may be self-administeredor administered by a health care professional.

Parameters that can be evaluated for determining a dose of thekallikrein inhibitor for systemic administration, are described belowwith regards to DX-88 (a non-naturally occurring kallikrein inhibitor,SEQ ID NO:2). The total amount of circulating prekallikrein in plasma isreported to be approximately 500 nM to 600 nM (Silverberg et al., “TheContact System and Its Disorders,” in Blood: Principles and Practice ofHematology. Handin, R. et al., eds, J B Lippincott Co., Philadelphia.1995). If all prekallikrein is activated, about 520 nmoles/L of DX-88(DX88) can be used to inhibit kallikrein in a stoichiometric manner. Anindividual having 5 L of plasma would require a dose of 2.6 micromolesDX-88, or approximately 18 mg based on the molecular weight of DX-88 of7,054 Daltons. This was calculated as follows: the K_(i) of DX88 is0.025 nM. When it is desired to have a concentration of plasmakallikrein (PK) of, e.g., 1 nM, the following formula for a tightbinding inhibitor indicates that the concentration of free DX-88 is 12.0nM. Thus, the total amount of DX-88 needed would be 499+12 or 511 nM.

$\left\lbrack {{DX}\; 88_{total}} \right\rbrack = {\frac{K_{i,{app}}\left\lbrack {{p\;{Kal}} - {{DX}\; 88}} \right\rbrack}{\left\lbrack {{DX}\; 88_{free}} \right\rbrack} + \left\lbrack {{p\;{Kal}} - {{DX}\; 88}} \right\rbrack}$511  n M = (0.025)(499)/(1) + (499)

The dose can be reduced proportionally if not all of the prekallikreinis activated or if a portion of the kallikrein is deactivated by anendogenous inhibitor, e.g., C1 esterase inhibitor (C1INH). Thus, incertain embodiments, about 5, 10, 15, 20, 30, 40, 60.80, 120, 250, 500,600, 700, 800, 1000 mg of DX-88 can be administered to a subject, in asingle dose or in one or more doses spread over a twenty-four hourperiod. Consideration of several other factors may provide a moreaccurate estimation of the dose of DX-88 required in practice, such aspatient age, weight, and severity of the condition 0.

In some embodiments, the kallikrein inhibitor polypeptide isadministered in a dose of about 1-500 mg/m², preferably about 1-250mg/m², 1-100 mg/m².

(G) Devices and Kits

Pharmaceutical compositions that include the kallikrein inhibitor can beadministered with a medical device. The device can designed withfeatures such as portability, room temperature storage, and ease of useso that it can be used in settings outside of a hospital or emergencyroom/urgent care facility (e.g., by the patient or a caregiver in thehome or in a doctor's office). The device can include, e.g., one or morehousings for storing pharmaceutical preparations that include anisolated kallikrein inhibitor, and can be configured to deliver one ormore unit doses of the agent or agents.

I.V. administration may be by bolus or infusion, using appropriateinjection or infusion devices (e.g., catheters, infusion pumps,implants, and the like). Subcutaneous injection may be as an infusion,for example using a catheter and infusion pump or implantable device.Many other devices, implants, delivery systems, and modules are alsoknown.

When the kallikrein inhibitor is distributed as a lyophilized powder, itmust be reconstituted prior to use. Manual reconstitution (e.g., manualaddition of diluent to the lyophilized formulation by injection throughan injection port into the container containing the lyophilizedformulation) may be used, or the kallikrein inhibitor may be provided ina device configured for automatic reconstitution (e.g., automaticaddition of the diluent to the lyophilized formulation), such as theBECTON-DICKINSON BD™ Liquid Dry Injector.

The isolated kallikrein inhibitor can be provided in a kit. In oneembodiment, the kit includes (a) a container that contains a compositionthat includes an isolated kallikrein inhibitor, and (b) informationalmaterial that relates to the methods described herein and/or the use ofthe agents for therapeutic benefit.

In certain embodiments, the kit includes also includes anothertherapeutic agent. For example, the kit includes a first container thatcontains a composition that includes the isolated kallikrein inhibitor,and a second container that includes the other therapeutic agent. Theisolated kallikrein inhibitor and the other therapeutic agent may besupplied in the same container for use in methods in which thekallikrein inhibitor and the therapeutic agent are administered as asingle composition.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods of administering the isolated kallikrein inhibitor, e.g., in asuitable dose, dosage form, or mode of administration (e.g., a dose,dosage form, or mode of administration described herein), to treat asubject ( ). The information can be provided in a variety of formats,include printed text, computer readable material, video recording, oraudio recording, or a information that provides a link or address tosubstantive material.

In addition to the isolated kallikrein inhibitor (and, if present, theadditional therapeutic agent(s)), the composition in the kit can includeother ingredients, such as a solvent or buffer, a stabilizer, or apreservative. The isolated kallikrein inhibitor (and other therapeuticagent, if present) can be provided in any form, e.g., liquid, dried orlyophilized form, preferably substantially pure and/or sterile. When theagents are provided in a liquid solution, the liquid solution preferablyis an aqueous solution. When the agents are provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent. e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the isolated kallikrein inhibitor and anothertherapeutic agent, e.g., in a desired ratio. For example, the kitincludes a plurality of syringes, ampoules, foil packets, blister packs,or medical devices, e.g., each containing a single combination unitdose. The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty, but suitable for loading.

EXAMPLES

The following examples provide further illustration and are notlimiting.

Example 1: Association Between Cleaved High Molecule Weight Kininogen(HMWK) and Autoimmune Diseases

To determine the levels of cleaved HMWK in autoimmune diseases, theamounts of intact and cleaved HMWK in plasma samples obtained frompatients of rheumatoid arthritis (RA), ulcerative colitis (UC), andCrohn's disease (CD), as well as from healthy subjects, were measuredusing Western blot with LiCor detection as described below.

(i) Sample Preparation

The treated HMWK-deficient plasma was prepared by adding 10 μL of 10×anti-protease inhibitor cocktail to 90 μL 100% HMWK-deficient plasma.The solution was allowed to sit for at least 30 minutes prior to use.

A 1:4 intermediate of the single-chain HMWK was prepared by adding 5 μLof the stock solution (1.61 mg/mL) to 15 μL of treated HMWK-deficientplasma. A 1:4 intermediate of the two-chain HMWK was prepared by adding5 μL of the stock solution (2.01 mg/mL) to 15 μL of treatedHWMK-deficient plasma.

A 45 μg/mL treated HMWK-deficient control plasma solution was preparedby adding 3.35 μL of the 1:4 single-chain HMWK intermediate and 2.69 μLof the 1:4 two-chain HWMK intermediate to 23.96 μL of treatedHWMK-deficient plasma.

Each sample was diluted to 5% plasma (1:20) in 1×TBS by adding 5 μL ofthe plasma sample to 95 μL of 1×TBS. The non-reduced samples wereprepared by adding 5 μL of 4× sample buffer to 15 μL of 5% sample. Thereduced samples were prepared by adding 5 μL of the 4× sample buffer and2 μL of 10× reducing agent to 13 μL of 5% sample.

All of the samples were heated at 95° C. for 5 minutes using a heatblock. Each sample was briefly centrifuged to remove any solution fromthe cap of the sample tubes.

(ii) Gel Loading, Running, and Transfer

Tris-Acetate running buffer was prepared by adding 100 mL of 20× runningbuffer to 1.900 mL of DI water. A volume of 4 μL of the one-colorprotein marker was used as a control in all assays. A volume of 13 μL ofnon-reduced samples and the reduced samples was added to lanes of a gel,of gel 1. The gels were run at 125 volts for −2 hours.

The iBlot Filter Paper was placed in DI water and soaked for 5 minutes.The Anode Stack, Bottom was unsealed and placed on the blotting surfaceof the iBlot with the copper side down. Upon completion of the gel runs,two gel cassettes were opened and the gels removed. The gels were placedonto the transfer membrane. The pre-soaked filter paper was placed ontop of the gels. The Cathode Stack, Top was unsealed and placed on topof the filter paper, with the copper side up. The bubbles in the stackwere gently rolled out using the blotting roller. The disposable spongewas placed into the iBlot lid. The iBlot was turned on and program P0was selected.

Upon completion of the iBlot transfer, opened the iBlot and discardedthe sponge, cathode stack, and gels. Each membrane was individuallyremoved from the iBlot and placed it into a plastic tray containing 20mL of Odyssey Blocking Buffer. The membranes were incubated on a plateshaker at room temperature for 1 hour.

(iii) Westernblot Assay with LiCor Detection

A 1 μg/mL primary antibody solution was prepared by adding 57.14 μL ofMouse antiLC HMWK, clone #11H05 (stock concentration 1.4 mg/mL) to 80 mLof Odyssey Blocker+0.2% TWEEN® 20 (Polysorbate 20). The blocking bufferwas removed from the plastic trays. A volume of 20 mL of primaryantibody solution was added to each tray and the membranes wereincubated on a plate shaker at room temperature for 1 hour. The Goatanti-mouse IgG IRDye 680 solution was prepared at a 1:15,000 dilution byadding 5.33 μL of the stock solution to 80 mL of Odyssey Blocker+0.2%TWEEN® 20 (Polysorbate 20). The primary antibody solution was removedfrom the plastic trays. The membranes were washed with 20 mL 1×PBS+ 0.1%TWEEN® 20 (Polysorbate 20) for five minutes and then the wash wasdiscarded. Repeated 3 times for a total of 4 washes. A volume of 20 mLof the Goat anti-Mouse IgG IRDye 680 solution was added to each tray.The trays were covered with aluminum foil to protect the membranes andthe secondary antibody solution from light. The membranes were incubatedon a plate shaker at room temperature for 1 hour. The secondary antibodysolution was removed from the plastic trays. The membranes were washedwith 20 mL 1×PBS+0.1% TWEEN® 20 (Polysorbate 20) for five minutes andthen the wash was discarded. Repeated 3 times for a total of 4 washes.The membranes were washed with PBS and scanned on the LiCor Odyssey CLx.The membranes were covered with aluminum foil and allowed to dryovernight. The dried membranes were placed in a protective cover sheetand saved for later use.

(iv) Results:

The control, treated HMWK-deficient plasma spiked with 45 μg/mL ofsingle and two-chain HMWK, performed as expected producing bandingaround 120 and 95 kDa. The normal human plasma produced bands around 120and 100 kDa with 21.3% of the HMWK cleaved. Patient samples containinganti-protease inhibitor cocktails produced dramatically differentresults than patient samples collected in sodium citrate. The patientsamples collected with anti-protease inhibitor added containedsignificantly more single-chain HMWK than reduced two-chain HMWK. Thisresult indicates that the addition of anti-protease inhibitor cocktailsis necessary to prevent the cleavage of HMWK after collection.

Plasma samples from a number of UC, CD, RA, and HAE patients wereexamined in this study. The control sample and normal human plasmaproduced the expected results. The HAE attack sample contained morecleaved HWMK than the HAE basal sample. As shown in Table 1 below andalso in FIG. 1, a large majority of the samples from the autoimmunepatients, especially the rheumatoid arthritis samples, only producedbanding at 46 kDa, indicating that the level of cleaved HWMK isassociated with autoimmune diseases, such as RA, UC, and CD. Morespecifically, RA flares were found to be associated with extensivecleaved HMWK; Crohn's disease was found to has a moderate amount ofcleaved HMWK; and ulcerative colitis was found to have a moderate amountof cleaved HMWK.

In sum, the results of this study indicate contact activation inautoimmune diseases such as CD, UC, and RA plasma. Accordingly, an agentthat inhibits contact activation, such as an inhibitor of pKal (e.g.,those described herein) can be effective in treating such diseases.Further, the results of this study indicate that the level of cleavedHMWK can serve as a reliable biomarker for identifying patients havingor at risk for autoimmune diseases such as CD, UC, and RA and/or as abiomarker for assessing the efficacy of an treatment for such anautoimmune disease.

TABLE 1 Levels of cleaved HMWK in RA, UC, and CD Patients Disease %Cleaved UID Age Gender Medications Diagnosis Stage HMWK 106659 34 MalePentasa 500 mg Crohn's Disease, High Flare 3 cholesterol 104006 50Female Pentasa 500 mg Crohn's Disease Stable 12.2 100929 59 MaleCiproflaxacin, Flagyl Crohn's Disease Stable 32.7 118166 62 MaleTinidazole 500 mg Crohn's disease Stable 10.3 105772 22 Female LialdaCrohn's disease Stable 95.7 72211 53 Female Canasa suppositories,Vitamin C Ulcerative Colitis Stable 13.1 96319 78 Male Asacol, Canasa,Hydrocortisone, Ulcerative colitis Stable 4.4 Prilosec, Tylenol, ASA,Diltazem, Lorazepam, Align probiotic, Vitamins 93587 24 Female Canasasuppositories, Carafate, Ulcerative colitis Stable 100 Omeprazole,Asacol, Remicade 92122 21 Female Prednisone, Lialda, Remicade,Ulcerative colitis Stable 4.9 Percocet 94441 77 Male Lialda, PlavixUlcerative colitis Flare 16.5 147603 72 Female Enbrel, Arava 20 mg,Folic acid Rheumatoid Arthritis (flare), Flare 100 1 mg, Azelphadine 500mg, 4 out of 5 patients at 100% Synthroid .175 mg, 100953 50 FemalePlaquenil, Meloxicam, MTX, Rheumatoid Arthritis (RA), Flare 100Prednisone Hypertension (HTN), Bursitis 72015 36 Female Methotrexate;Folic acid; MVI; Rheumatoid Arthritis (RA) Flare 100 Calcium and vitaminD; Prednisone 31567 49 Female Enbrel; Tylenol; Medrol; Prilosec;Rheumatoid Arthritis (RA) Flare 100 Citalopram; HCT; Fish oil; Naprosyn33503 44 Female Synthroid; Orencia; Prednisone; Rheumatoid Arthritis(RA) Flare 46.2 Ambien; Methotrexate; Hydrocodone; Folic acid

REFERENCES

The contents of all cited references including literature references,issued patents, published or non-published patent applications citedthroughout this application as well as those listed below are herebyexpressly incorporated by reference in their entireties for the purposesor subject matter referenced herein. In case of conflict, the presentapplication, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the present disclosure described herein. The scope of thepresent disclosure is not intended to be limited to the abovedescription, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The present disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thepresent disclosure includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process.

Furthermore, the present disclosure encompasses all variations,combinations, and permutations in which one or more limitations,elements, clauses, and descriptive terms from one or more of the listedclaims is introduced into another claim. For example, any claim that isdependent on another claim can be modified to include one or morelimitations found in any other claim that is dependent on the same baseclaim. Where elements are presented as lists, e.g., in Markush groupformat, each subgroup of the elements is also disclosed, and anyelement(s) can be removed from the group. It should it be understoodthat, in general, where the present disclosure, or aspects of thepresent disclosure, is/are referred to as comprising particular elementsand/or features, certain embodiments of the present disclosure oraspects of the present disclosure consist, or consist essentially of,such elements and/or features. For purposes of simplicity, thoseembodiments have not been specifically set forth in haec verba herein.It is also noted that the terms “comprising” and “containing” areintended to be open and permits the inclusion of additional elements orsteps. Where ranges are given, endpoints are included. Furthermore,unless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the present disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the present disclosure can be excluded from anyclaim, for any reason, whether or not related to the existence of priorart.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present disclosure, as defined in the following claims.

What is claimed is:
 1. A method of treating an autoimmune disease in asubject, the method comprising administering to a subject in needthereof an effective amount of a plasma kallikrein (pKal) inhibitor, ananti-inflammatory agent, or a combination thereof, wherein the subjecthas an elevated level of cleaved high molecular weight kininogen (HMWK)as compared to a healthy subject in one or more biological samples ofthe subject, wherein the one or more biological samples are bloodsamples, serum samples, or plasma samples; wherein the subject is ahuman subject having or suspected of having the autoimmune disease,which is rheumatoid arthritis (RA), Crohn's disease (CD), or ulcerativecolitis (UC).
 2. The method of claim 1, wherein the subject isadministered a pKal inhibitor.
 3. The method of claim 2, wherein thepKal inhibitor is a Kunitz domain polypeptide or an antibody binding topKal.
 4. The method of claim 2, wherein the pKal inhibitor is DX-2930,DX-88, or EPIKAL-2.
 5. The method of claim 1, wherein the subject isadministered an anti-inflammatory agent, which is a steroid or anonsteroidal anti-inflammatory drug (NSAID).
 6. A method of treating anautoimmune disease, the method comprising: (i) identifying a subject ashaving or at risk for an autoimmune disease based on the level ofcleaved high molecular weight kininogen (HMWK), wherein the subject hasan elevated level of cleaved HMWK as compared to a healthy subject, inone or more biological samples of the subject; and (ii) administering tothe subject an effective amount of a plasma kallikrein (pKal) inhibitor,an anti-inflammatory agent, or a combination thereof, wherein the one ormore biological samples are blood samples, serum samples, or plasmasamples; wherein the subject is a human subject having or suspected ofhaving the autoimmune disease, which is rheumatoid arthritis (RA),Crohn's disease (CD), or ulcerative colitis (UC).
 7. The method of claim6, wherein the subject is administered a pKal inhibitor.
 8. The methodof claim 7, wherein the pKal inhibitor is a Kunitz domain polypeptide oran antibody binding to pKal.
 9. The method of claim 8, wherein the pKalinhibitor is DX-2930, DX-88, or EPIKAL-2.
 10. The method of claim 6,wherein the subject is administered an anti-inflammatory agent, which isa steroid or a nonsteroidal anti-inflammatory drug (NSAID).